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Assessment of the scavenging activity of crude methanolic stem bark extract of Newbouldia Laevis on selected free radicals.

Introduction

Free radicals are chemical entities characterized by a high reactivity. Varying reactivities notwithstanding, radicals, free radicals inclusive have been known to be generally less stable than non-radicals (Paolo et al, 1991). Free radical formation during the metabolism of xenobiotics is therefore an important mechanism employed by toxic agents in causing cellular damage. Reactive oxygen species (ROS) capable of damaging DNA, proteins, carbohydrates and lipids are generated in aerobic organisms. These ROS include superoxide anion radical, hydrogen peroxide, hydroxyl radical, and single molecular oxygen. The effects of these ROS are controlled by a system of enzymic and non- enzymic antioxidants. These antioxidants eliminate pro oxidants and scavenge free radicals (Arouma, 1996).

Oxygen is essential and central in free radical pathology due to its physicochemical properties such as water solubility and relatively high electron negativity (Tappel, 1996). Free radicals, especially the oxygen radical, superoxide, when formed could lead to the formation of other radicals. In fact, the toxicity of [O.sup.[??].sub.2] in living organisms is due to its conversion into [OH.sup.[??] and into reactive radical -metal complexes. The process through which superoxide and hydrogen peroxide are converted into [OH.sup.[??] and into reactive radical complexes are either referred to as the iron-catalyzed Haber- Weiss reaction or the superoxide driven Fenton reaction (Paolo et al., 1991; Goldstein & Meyerstein, 1993; Fenton, 1894; Koppenol, 1993).

Numerous research have been carried out on various parts of N. laevis (Bignoniaceae). N. laevis, known more commonly as African Border tree is used for therapeutic purpose against a number of diseases. In Ivory Coast and Nigeria, stem bark decoctions of N. laevis is used for the treatment of epilepsy and convulsions in children. After pulping up to a paste, the bark is used for treatment of rheumatism, especially painful arthritis of the knees in Senegal. In Nigeria, decoctions of leaves and roots made from boiling are used as febrifuge. Treatment of breast tumors with bark and leaf decoctions is common in Ghana and Nigeria (Burkill, 1985; Burkill, 1997). Extracts of all parts of N. laevis, that is, leaves, stem bark and root have been shown to exhibit antimicrobial activity (Ogunlana & Ramstad, 1975; Kuete et al, 2007; Hounzangbe-Adote et al, 2005; Gbaessor et al, 2006; Eyong et al, 2006). Sedative effects of the methanolic leaf extract of N. laevis in mice and rats have also been studied and reported (Amos et al, 2002).

Materials and methods

Extraction of plant materials

N. laevis stem bark was collected from Amina way, University of Ibadan, Oyo state, Nigeria. Stem bark sample was authenticated at the Herbarium, Botany Department of the institution, and sample portions were deposited at the herbarium. Stem bark was air--dried in shade and powdered. 150g of powdered plant stem bark was extracted in 900m1, 95% v/v methanol by maceration for 48 hours. After decantation of crude extract, filtration and concentration was carried out using Rotary Evaporator, and the weight of concentrated dried stem bark obtained was recorded for the calculation of yield.

Determination of Hydrogen Peroxide [H.sub.2][O.sub.2] (scavenging activity of plant extract)

Hydrogen Peroxide scavenging activity of plant extract was determined by the method of Ruch et al (1989) by Gow--Chin and Hui--Yin (1995). 20 -400[micro]g Buthylated hydroxyanisole (BHA) was added in place of plant extract in 4m1 distilled water and the solution was added to 0.6m1 [H.sub.2][O.sub.2] solution in PBS. Absorbance was determined 10 minutes later against a blank solution similar to that above.

Superoxide scavenging activity of plant extract

The effect of plant extract on superoxide generated in a non enzymic system was measured spectrophotometrically using the method of Gow-Chin and Hui-Yin (1995). The reaction mixture consisted of (10 - 1000[micro]g) dilutions of plant extract made to lml with distilled water, lml, 60[micro]M phenazine methosulphate (PMS), in phosphate buffer (O.1M, pH 7.4) and 150[micro]M, lml nitrroblue tetrazolium (NBT) in phosphate buffer. Incubation at ambient temperature followed for 5minutes, and the resultant colour was read spectrophotometrically at 560nm against a blank. The effect of Buthylated Hydroxyanisole (BHA) was also determined by replacing plant extract with lml BHA (10 - 1000[micro]g) in methanol in the reaction mixture.

Determination of the effect of plant extract on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical.

1mM DPPH solution was prepared by dissolving 31.54 mg DPPH in 95% v/v methanol and made up to 50m1 with same. DPPH scavenging activity was assessed using the method of Hatano et al., (1998) as modified by Gow-Chin Yen and Hui-Yin Chen (1995). Absorbance of the resulting solution was measured spectrophotometrically at 517nm. The effect of BHA on DPPH was also assessed for comparism with that of plant extract. Methanolic dilutions (0.2, 0.4, 0.6, 0.8, 1.0ml) of lmg/ml BHA was made to 4m1 with distilled water. lml DPPH radical (1nM) was added to each tube, and same procedure as in DPPH scavenging experiment was followed.

Statistical analysis was carried out using T-test (Paired two-sample for means). Values were considered significant for [T.sub.crit][??] < [T.sub.calc][??} at 95% confidence limit (P < 0.05)

Result and Discussion

The extraction of 150g plant material in 900m1 (95%v/v) methanol yielded o.3178g of material after extraction. Percentage yield was therefore 4.2117%. Table 1 show data on the scavenging activity of plant extracts on hydrogen peroxide.

Percentage free radical scavenging activity was calculated using the formula:

% SA = ([A.sub.c-[A.sub.E]) [??] 100 Ac

Where [A.sub.c] = Absorbance of control [A.sub.E] = Absorbance of control % SA = Percentage scavenging activity

Plant extract scavenged [H.sub.2][O.sub.2] more effectively than BHA (Figure 1). Both BHA and plant extract scavenged H,O, in concentration--dependent manner. Interestingly, at 0.0217mg/ml, both plant extract and BHA scavenged hydrogen peroxide to the same extent, but at 0.0435mg/ml, plant extract appeared to be about 37.5% more efficient than BHA at scavenging hydrogen peroxide.

Superoxide anion scavenging data shown in Table 2 and corroborated by Figure 2 present an interesting trend. Even at the lowest of the concentrations used (0.05mg/ml), plant extract scavenged 81.48% of superoxide anion generated in the system while BHA only mopped up 5.82% of same, a display of a scavenging efficiency of 1300% over BHA. Figure 3 (percentage DPPH scavenging activity) shows a trend that is removed from that observed for hydrogen peroxide and superoxide anion above. Though both plant extract and BHA scavenged DPPH in concentration -dependent manner, BHA displayed better DPPH scavenging efficiency over plant extract. In fact, at the lowest concentration, BHA scavenges DPPH at twice the efficiency of plant extract. This may have been due to BHA's possession of a methoxy group which increases the accessibility of the radical centre of DPPH to BHA1 Indirect stimulation of lipid oxidation by superoxide as a result of superoxide and hydrogen peroxide act as precursors of singlet oxygen and hydroxyl radical (Cohen & Heikkila, 1974).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Statistical analysis (test of significance) of the data obtained from the free radical scavenging activity of plant extract and BHA using T-test (Paired two sample for means) showed that the difference between the free radical scavenging activities of plant extract and BHA on the natural ROS used, was significant (P<0.05). However, a non significant difference in DPPH scavenging activity was seen between the two at same confidence limit. In other words, the plant extract's superoxide radical and hydrogen peroxide scavenging activities were significantly higher than that of BHA. Although BHA's DPPH scavenging activity is higher than that of plant extract, the difference in the DPPH scavenging activity of the two was not significant (P<0.05).

[FIGURE 3 OMITTED]

Discussion

The ability of hydrogen peroxide to initiate lipid peroxidation is dependent on its ability to generate hydroxyl radical through the Fenton reaction (Namiki, 1990; Kellog, 1988). The ability of plant extract to scavenge [H.sub.2][O.sub.2] could also reflect its ability to inhibit the formation of hydroxyl radical in vivo. Harber Weiss reaction explains that both hydrogen peroxide and superoxide radical are required in the presence of metal catalyst for the formation of hydroxyl radical, the oxygen species largely responsible for the damage of macromolecules, the ability of plant extract to scavenge both hydrogen peroxide and superoxide anion to a remarkable extent would culminate in remarkable inhibition of hydroxyl radical formation, hence protection of macromolecules from oxidative damage.

BHA's better efficiency at scavenging DPPH may have been due to its possession of a methoxy group which increases the accessibility of the radical centre of DPPH to BHA (Magassouba et al, 2007). From Figures 1 to 3, a few deductions could be made. The [IC.sub.50], of N. laevis scavenging activity on hydrogen peroxide is 0.036mg/ml while the extract's [IC.sub.50], for its scavenging activity on both [SO.sub.2[??]] and DPPH could not be determined within the extract concentrations used for the experiment. The minimum plant extract concentration (0.05mg/ml) used for [SO.sub.2[??]] scavenging experiment had above the 50% scavenging effect, required to be measured for [IC.sub.50] determination. Conversely, the maximum plant extract concentration used for DPPH scavenging experiment did not scavenge up to 50% DPPH. [IC.sub.50] for hydrogen peroxide scavenging activity of BHA was 0.055mg/ml, while that of its DPPH scavenging activity could not be determined from the graph because its DPPH scavenging activity at the lowest concentration used in the experiment was more than 50%. Not many publications of research work done on the free radical scavenging activity of N. laevis are known yet.

This research provides information which could trigger further research in the direction of partial or full isolation and characterization of the constituents of leaf extract of N. laevis in order to decipher the specific phytochemical constituent(s) responsible for the free radical scavenging activity of the plant. When this is done, extracts of N. laevis could find important application in phytotherapy. Experiments confirming these activities of the extract of N. laevis in an in vivo system would be necessary. If the results of such experiments agree significantly with our in vitro results, then it would be easy to make the following inferences:

N. laevis extract would prevent oxidative damage to macromolecules by the following mechanisms among others:

* scavenging hydrogen peroxide

* scavenging superoxide anion radical

* inhibiting the formation of hydroxyl radical from the Fenton reaction since it scavenges hydrogen peroxide

* inhibiting the formation of hydroxyl radical from both hydrogen peroxide and superoxide as powered by Harber--Weiss reaction

References

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Corresponding Author: O.E. Ogunlana, Department of Biological Science, Covenant University, Canaan land, 10 Idi Iroko road, Ota, Ogun state, Nigeria. For correspondence: seyio2001@yahoo.com,

O.E. Ogunlana, Olubanke Ogunlana, O.E. Farombi: Assessment of the Scavenging Activity of Crude Methanolic Stem Bark Extract of Newbouldia Laevis on Selected Free Radicals: Adv. in Nat. Appl. Sci., 2(3): 249-254, 2008

(1) O.E. Ogunlana, (1) Olubanke Ogunlana, (2) O.E. Farombi

(1) Department of Biological Science, Covenant University, Canaan land, 10 Idi Iroko road, Ota, Ogun state, Nigeria.

(2) Department of Biochemistry, University of lbadan, Oyo state, Nigeria.
Table 1: Hydrogen Peroxide Scavenging Activity of Plant Extract.

Conc. of plant
extract Absorbance * (230nm)

(mg/ml) N. laevis BHA

0.0109 0.039 [+ or -] 0.001 0.041 [+ or -] 0.001
0.0217 0.036 [+ or -] 0.000 0.036 [+ or -] 0.002
0.0326 0.032 [+ or -] 0.000 0.035 [+ or -] 0.001
0.0435 0.019 [+ or -] 0.002 0.028 [+ or -] 0.001
0.0543 0.017 [+ or -] 0.002 0.024 [+ or -] 0.003

* Absorbance of control = 0.052 [+ or -] 0.001

Table 2: Superoxide Scavenging Activity of Plant Extract

Conc. of plant Absorbance * (560nm)
extract

(mg/ml) N. laevis BHA

0.0109 0.035 [+ or -] 0.002 0.178 [+ or -] 0.002
0.0217 0.018 [+ or -] 0.002 0.171 [+ or -] 0.002
0.0326 0.014 [+ or -] 0.003 0.170 [+ or -] 0.002
0.0435 0.005 [+ or -] 0.001 0.146 [+ or -] 0.005
0.0543 0.003 [+ or -] 0.000 0.142 [+ or -] 0.001

* Absorbance of control = 0.189 [+ or -] 0.003

Table 3: Dpph Scavenging Activity of Plant Extract.

Conc. of plant
extract Absorbance * (517nm)

(mg/ml) N. laevis BHA

0.04 0.613 [+ or -] 0.003 0.316 [+ or -] 0.012
0.08 0.592 [+ or -] 0.017 0.302 [+ or -] 0.002
0.12 0.548 [+ or -] 0.001 0.158 [+ or -] 0.031
0.16 0.522 [+ or -] 0.013 0.126 [+ or -] 0.001

* Absorbance of control = 0.725 [+ or -] 0.002
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Title Annotation:Original Article
Author:Ogunlana, O.E.; Ogunlana, Olubanke; Farombi, O.E.
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Date:Sep 1, 2008
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