MATERIALS AND METHODS

Materials
The studies were conducted in Lagos, Nigeria in 2006/2007. The leaves of M. barteri were collected at Ibadan, in Oyo, Nigeria and were authenticated by Mr. K.I. Odewo of Forest Research Institute of Nigeria (FRIN) Ibadan, Nigeria after comparing with voucher specimen with number FHI 107230. The leaves were dried at 30°C and hand crushed to fine powder.

Chemicals
Folin-Ciocalteu phenol reagent, gallic acid, anhydrous sodium carbonate, ascorbic acid, linoleic acid, ammonium thiocyanate, 2,6-Di-tert-buytl-4-methylphenol (BHT), 90% 1,1-diphenyl-2-picrylhydrazyl (DPPH), FeCl₂ tetrahydrate, anhydrous ferric chloride, potassium ferricyanide, [4,4’- [3-(2-pyridinyl-1,2,4-triazine-5,6-diyl]bisbenzenesulfonic acid] (ferrozine) and trichloroacetic acid were purchased from Sigma Chemical Company (St. Louis, MO). All other chemicals were of analytical grade BDH Chemical Laboratory (England, UK).

Preparation of Extract
About 500 g of powdered leaf was extracted with methanol (3 L) using Soxhlet apparatus for 48 h. The extract was concentrated under reduced pressure until a semi-solid sticky mass was obtained. The yield was 17.55% w/w.

Phytochemical Screening
The leaves of M. barteri were tested for the presence of alkaloid, anthraquinone, cardiac glycoside, steroidal nucleus, saponins, cyanogenetic glycoside and phenolic compounds using the method of Sofowora (1982).

Determination of Total Phenolic Content
The total phenolic content of the extract was determined with the Folin-Ciocalteu reagent (Mcdonald et al., 2001). The extract (100 mg mL^-1, 1.0 mL) was mixed with 5 mL Folin-Ciocalteu reagent (diluted ten-fold) and 4 mL (0.7 M) sodium carbonate. The mixture was allowed to stand for 1 h and the absorbance was measured at 765 nm. The analysis was carried out in triplicate and gallic acid was used as the standard for calibration curve. The total phenolic content was expressed as milligrams of gallic acid equivalents per gram of methanolic extract.

Measurement of DPPH Radical Scavenging Activity
The method of Koleva et al. (2002) with some modifications was used for the determination of free radical scavenging activity of the extract. The extract (0.02-0.75 mg mL^-1) of 1.0 mL was diluted with 4.0 mL of methanol and mixed with 1.0 mL of methanolic solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) (1 mM). The mixture was shaken immediately and allowed to stand at 20°C in dark for 30 min. The decrease in absorbance at 517 nm was then measured using spectrophotometer. BHT and ascorbic acid were tested and used as reference standards. All analysis was performed in triplicate and the ability to scavenge the DPPH radical was calculated using the following equation:

Evaluation of Reducing Ability
The reducing power of the extract was determined according to the method of Yen and Chen (1995) with some modifications. The extract (1.0 mL, 0.1-1.5 mg mL^-1) was mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%) was added then the mixture was incubated at 50°C for 20 min. A portion (2.5 mL) of trichloroacetic acid (10%) was added to the mixture and centrifuged at 1000 g for 10 min. The upper layer of the solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl₃ (0.5 mL, 0.1%) and the absorbance was measured at 700 nm against a blank in the spectrophotometer. Ascorbic acid and BHT were used as positive controls. Higher absorbance of the reaction mixture indicated increased reducing power.

Measurement of Ferrous Ion Chelating Capability
The Fe²⁺-chelating power was measured using the method of Decker and Welch (1990) with some modifications. The extract (1 mL, 0.1-10.0 mg mL^-1) was mixed with 4 mL of deionised water then treated with FeCl₂ (0.1 mL, 2.0 mM) and ferrozine (0.2 mL, 5 mM) The mixture was shaken and left to stand at 20°C for 20 min. The absorbance of the final solution was determined at 562 nm. EDTA was used as positive control and ability to chelate ferrous ion was determined using the following equation:

Antioxidant Activity
The antioxidant activity was measured using the method of Chang et al. (2002) with modifications. The extract (0.5 mL, 0.5-7.5 mg mL^-1) was mixed with linoleic acid emulsion (2.5 mL, 0.56% w/v, pH 7.0) and phosphate buffer (2 mL, 0.2 M, pH 7.0) and incubated at 60°C in the dark for 12 h to accelerate oxidation. Ferrous chloride solution and ammonium thiocyanate were added to 0.1 mL solution of the sample in sequence and the absorbance was read at 500 nm to determine the peroxide value. Ascorbic acid and BHT were used as positive controls. The solution without the extract or the controls was used as blank. The inhibitory effect of the extract in the linoleic acid system was determined as follows:

Statistical Analysis
All data were expressed as mean±standard deviation. Analysis of variance was performed by ANOVA procedures and p<0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION

The results of phytochemical investigation of the leaves of M. barteri showed presence of saponins and phenolic compounds. However, alkaloids, anthraquinones, cardiac and cyanogenetic glycosides were not detected. The total phenolic content of leaf extract of M. barteri was found to be 48.88 mg g^-1, as gallic acid. Phenolic compounds in plants have been reported to play a significant role in scavenging effect and prevention of lipid peroxidation due to the presence of hydroxyl moiety (Kahkonen et al., 1999). They may be responsible for the antioxidant activities observed in the tested extract.

Fig. 1:	Scavenging effects of M. barteri, Ascorbic acid and BHT on DPPH radical. Each value represents mean±standard deviation (n = 3)

Fig. 2:	Reducing power of M. barteri, Ascorbic acid and BHT. Each value represents mean±standard deviation (n = 3)

The radical scavenging effect of the extract and positive controls were concentration dependent (Fig. 1). The scavenging effects increased with increasing concentration then level off with further increase in concentration. The radical scavenging activity of the extract and positive controls decreased in the following order: ascorbic acid > BHT > M. barteri with IC₅₀ of 0.10, 0.15 and 0.18 mg mL^-1, respectively. DPPH radical scavenging activities have reported to be due to proton donating ability of the antioxidant (Yamagushi et al., 1998; Mokbel and Hashinaga, 2006) thus the antioxidant activities of the extract may be attributed to its hydrogen donating power. The radical scavenging activity of the extract is comparable to that of BHT and ascorbic acid and no significant difference between them p>0.05.

Fig. 3:	Chelating effect of M. barteri leaf extract on Fe2+ ion. Each value represents mean±standard deviation (n = 3)

Fig. 4:	Effects of M. barteri leaf extract on linoleic acid peroxidation. Each value represents mean±standard deviation (n = 3)

The reducing activities increase with increasing concentration of the antioxidant (Fig. 2). Reduction of Fe³⁺/ferricyanide complex to the ferrous form is commonly used for the assessment of antioxidant properties of compounds. The reducing power of these compounds is due to their ability to donate electrons (Yen and Chen, 1995; Kahkonen et al., 1999). The results obtained showed M. barteri leaf extract as a reducing agent that could react with free radicals and convert them to more stable compounds. The reducing power of the extract of M. barteri and standard compounds follow the order of ascorbic acid > BHT > M. barteri.

The chelating ability also increased with increase in leaf extract concentration and reached 30.5% at concentration of 0.5 mg mL^-1 then decreased as the concentration increased (Fig. 3). EDTA showed better chelating power (IC₅₀ = 0.075 mg mL^-1) than the extract at all tested concentration. Iron chelating power is essential in antioxidant properties because it reduces the production of oxyradicals and lipid peroxidation thus protecting against oxidative damage (Willet, 1995; Nice, 1997; Gulcin et al., 2003). The results obtained showed that metal chelation plays very little role in antioxidant properties of the extract.

Lipid peroxidation involves oxidation of polyunsaturated fatty acid in the cell membrane (Fig. 4) and it was reported to be responsible for cell membrane destruction and cell damage (Yoshikawa et al., 1997). The leaf extract of M. barteri inhibited lipid peroxidation in a dose dependent manner but its activity was significantly less (p<0.05) than that of BHT and ascorbic acid. The result suggested that the extract could be used in treatment of free radicals and oxidative damage induced diseases. The antioxidant capacity increased in the order macaranga < ascorbic acid < BHT with IC₅₀ 10.21, 6.41 and 4.12 mg mL^-1, respectively.

We may conclude that the M. barteri leaf extract has a potent antioxidant property. Although the activity is relatively lower than that of BHT and ascorbic acid, the extract may be a veritable source of bioactive compounds with better activities after fractionation.

ACKNOWLEDGMENTS

Authors are grateful to Mr. Adeleke and Mr. Ojobo of University of Lagos for their technical assistance.