ANTIOXIDANT ACTIVITY AND PHYTOCHEMISTRY STUDY OF COMBRETUM OBANENSE STEM (BAK. F) HUTCH. AND DALZ

Objective: The objective of the present study was designed to test the antioxidant activity and phytochemical property of the methanol/ methylene chloride (1:1) extract of Combretum obanense. Place and Duration of Study: The International Centre for Ethnomedicine and Drug Development (InterCEDD), 110 Aku Road Nsukka, Enugu State, Nigeria, between January 2014 and July 2014. Methodology: We had assessed methanol/methylene chloride (1:1) stem extract of C. obanense, for antioxidant potentials with the help of DPPH (1, 1-diphenyl-2-picryl hydrazyl) free radical scavenging, reducing power assay and phytochemical analysis.


INTRODUCTION
Combretum is a rainforest shrub, flower spikes up to 5 cm long.Leaves are glabrous and glandular beneath with pits in the axils of the main nerves, lamina elliptic, coriaceous, 8 -12 cm long and about 4 cm broad.Main lateral nerves are prominently looped.It is widely distributed and native to tropical and southern Africa [1], about 5 species Madagascan, some 25 species to tropical Asiaand approximately 210 species tropical America.The genus is absent in Australia [2].Several species of Combretum are used in African or Indian herbal medicine.The class of chemical compound known as Combretastatins were isolated from South African Bushwillow (Combretum caffrum), from which they get their name.One synthetic derivative, fosbretabulin disodum (combretastatin A4 phosphate) underwent preliminary study for the treatment of anaplastic thyroid cancer [3], but it was not effective enough to progress to more advanced trial.The species of Combretum molle is also recorded to contain antioxidants such as punicalagin, which is also found in pomegranates (Punicagra natum), a somewhat related plant.From three results of the antimicrobial screening of some species of Combretum such as Combretum fragrans, and Combretum padoides, the methanolic extracts of these species showed marked inhibition against Gram-positive bacteria, and were also good inhibitors of Enterobacter aerogenes [4].Combretum extracts or isolates have shown in vitro bioactivities such as antibacterial, antifungal, anti-hyperglycemic, cytotoxicity against various human tumor cells lines, anti-inflammatory, anti-snake, anti-malaria, and anti-oxidant effect [5].At least twenty four species of Combretum are well known in African traditional medicine, and used for the treatment of a variety of ailments and diseases, ranging from scorpion and snake bites, mental problems, heart and worm remedies to fever and microbial infection [6 ]. C. obanense is a scandent shrub of the forest region.The leaves are glabrous and glandular beneath, with pits in the axils of the main nerves; lamina elliptic, coriaceous, 8-12 cm long, 3.5-5.5 cm broad; main

Phytochemical Analysis of the Extract
The methanol: methylene Chloride (1:1) extract of Combretum obanense was subjected to the following phytochemical tests described by Trease and Evans [8].

In vitro antioxidant tests 2.3.1 Test on reducing power model
The reducing power was determined by the method of Athukorala [9].In this model, 1.0 ml extract of different concentrations (25, 50, 100, 200 and 400 µg/ml) were each mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6).To each of the mixtures, 2.5 ml of potassium ferrocyanide (30 mM) was added and incubated at 50° C for 20 min.Thereafter, 2.5 ml of 0.6 M trichloroacetic acid (TCA) was added to the reaction mixtures and centrifuged for 10 min at 3000 rpm.The upper layer of each solution (2.5 ml) was decanted and mixed with 2.5 ml of distilled water and 0.5 ml of FeCl 3 (6 mM).The change in color was observed and then the absorbance of each mixture was measured at 700 nm using a UV-Vis Spectrophotometer.The same treatment was used for the different concentrations of the standard, and one for the normal control.Ascorbic acid was used as the positive control [10], with distilled water as the normal control.A higher absorbance of the reaction mixture indicated greater reducing power.

Test for free radical scavenging activity: DPPH assay
DPPH radical scavenging activity was measured using the methods of (Cotelle) [11] and (McCune and Johns) [12] with some modifications.The reaction mixture (3.0 ml) was prepared and it consisted of 1.0 ml of DPPH in methanol (100 µM), 1.0 ml of methanol and 1.0 ml each for the different concentrations of the extract and standard.The reaction mixtures were incubated for 10 minutes in a dark room, and then their absorbance were measured at 517nm using a UV-Vis Spectrophotometer.The positive control (standard) used was ascorbic acid (Blois) [13].The same treatment was given to the normal control that consisted of 1.0 ml of the 100 µM DPPH solution in methanol + 2ml of methanol, and the absorbance was determined.The percentage of inhibition was calculated using the formula: Where: A 0 is the absorbance of control A 1 is the absorbance of test.

The reducing power ability of C. obanense extract and ascorbic acid
Table 2 shows the results of the different absorbance values obtained from the reducing ability of C. obanense extract compared to that of the standard ascorbic acid at different concentrations.

The reducing power ability of Combretum obanense extract and ascorbic acid
Table 2 shows the results of the different absorbance values obtained from the reducing ability of C. obanense extract compared to that of the standard ascorbic acid at different concentrations.

The DPPH free radical scavenging assay
Table 3 indicates the absorbance values and percentage inhibition of different concentration of ascorbic acid and C. obanense using DPPH free radical scavenging assay.All values were significant at p< 0.001.Antioxidant compounds act by several mechanism such as inhibition of generation and scavenging activity against reactive oxygen species (ROS); reducing power; metal chelation; activity as antioxidant enzymes; inhibition of oxidative enzymes, among others [14].It has been established that oxidative damage caused by reactive oxygen species (ROS) leads to DNA lesions, loss of functions of enzymes, increased cell permeability, disturbed signaling over the cell and eventual necrotic cell death or apoptosis [14].The aim of the present study was to evaluate the antioxidant potential of C. obanense using the DPPH scavenging assay and Reducing Power Assay Model.DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule which is widely used to investigate radical scavenging activity.In DPPH radical scavenging assay, antioxidants react with DPPH (deep violet color) and convert it to yellow coloured α,αdiphenyl-β-picryl hydrazine.The degree of discoloration indicates the Radical-scavenging potential of the antioxidant [15,16].
The extract was capable of neutralizing the DPPH free radicals via hydrogen donating activity by 78.09, 80.13, 80.54, 80.45, and 86.99% at concentrations of 25, 50, 100, 200, and 400 µg/ml respectively.As shown in table3, DPPH scavenging was increased in a concentration dependent manner compared to ascorbic acid, which was used as the positive antioxidant control in this investigation.
The reducing ability of a compound generally depends on the presence of reductants [17] which have been exhibiting antioxidative potential by breaking the free radical chain and donating a hydrogen atom [18].The presence of reductants in extract causes the reduction of the Fe3+/ferricynide complex to the ferrous form.Therefore, the Fe2+ can be monitored by measuring the formation of Perl's Purssian blue at 700 nm.Table2 shows the reductive capabilities of the extract compared to ascorbic acid.Reducing power of the extract ranges from 0.7490 ± 0.0017 to 0.1647 ± 0.0007.This observation could be attributed to the difference in the levels of the phytochemicals in the extract which are responsible for metal reduction probably due to their highly nucleophilic nature that enables them to readily donate proton to electron deficient centers to cause reduction.

CONCLUSION
From the result obtained from the experiment it is concluded that extract of C. obanense has good antioxidant property.The finding supports the traditional use of this plant and suggests the presence of biological active components which may be worth further investigation and elucidation.

Table 1
shows the phytochemical constituents of C. obanense stem.

Table 2 : The reducing power ability of C. obanense extract and ascorbic acid
* Combretum obanense * * p < 0.01 (values compared to the control)