Free-radical Scavenging Activity

In cells, free radicals are continuously produced either as by-products of metabolism or deliberately as in phagocytes (Cheeseman and Slater 1993). The model of scavenging DPPH radical is especially useful in evaluating chain-breaking activity in the propagation phase of lipid (and protein) oxidation (Manzocco et al. 1998). The effect of antioxidants on DPPH radical scavenging was thought to be a result of their hydrogen donating ability (Gulcin et al. 2004). Figure 1 shows the dose-response curves for DPPH radical scavenging activities of five nanoemulsions the DPPH scavenging activity of the aqueous extracts of E. ulmoides increased with increasing amount of the extract. Extract from the leaf showed a significant (p < 0.05) stronger DPPH scavenging activity ((that is a lower [IC.sub.50] at 0.274 [+ or -] 0.010 mg/mL) than other extracts, and its polyphenolic content (112.79 [+ or -] 7.18 mg/g) was also significant higher than other extracts. Although the polyphenolic content of the extract of male flower was not significantly different from that of cortex, its DPPH scavenging effect (I[C.sub.50] - 0.596 [+ or -] 0.003 mg/mL) was significantly stronger than that of cortex (I[C.sub.50] - 0.955 [+ or -] 0.037 mg/mL). The order of DPPH scavenging activity was ascorbic acid > BHT > leaf> male flower > cortex > fruit.

Metal Chelating Effect

Metal chelating capacity is claimed as one of the antioxidant activity mechanisms (Diplock (1996, Yildirim et al. 2001), since it reduces the concentration of the catalyzing transition metal in lipid peroxidation (Hsu et al. 2003). It has been reported that chelating agents, which complex with metal by [sigma]-bonds, are effective as secondary antioxidants because they reduce the redox potential, thereby stabilizing the oxidized form of the metal ion (Gordon 1990). Ferrozine can quantitatively form complexes with [Fe.sup.2+]. In the presence of extract of E. ulmoides, the complex formation is disrupted with the result that the red color of the complex is decreased. As shown in Table 1, leaf extract and ascorbic acid exhibit significantly higher metal chelating effect than other extracts and BHT. In the concentration of 1 mg/mL, their metal chelating effects were 69.73 [+ or -] 2.12 percent and 67.70 [+ or -] 2.43 percent respectively. The extracts from fruit and cortex also demonstrated a certain ability to chelate metal ion. However, BHT and extract from male flower shows very poor (negligible) ferrous chelating ability. The decreasing order of the metal chelating effect was leaf > ascorbic acid > fruit > cortex > male flower and BHT.

Reducing Power

The reducing capacity of a compound may serve as an indicator of its potential antioxidant activity (Meir et al. 1995). In the reducing power assay, the antioxidant activity of samples was measured by their ability to reduce the [Fe.sup.3+]/ ferricyanide complex by forming ferrous products. Figure 2 shows the reductive power of E. ulmoides compared with BHT and ascorbic acid. Similar to the antioxidant activity, the reducing power of E. ulmoides increases with increasing dosage. All the extracts of the E. ulmoides showed a certain extent of reducing power. However, the reducing powers of BHT and ascorbic acid were found to be significantly more pronounced than that of E. ulmoides. Among the plant parts tested, the leaf extract and male flower extract exhibited greater reducing power than that of cortex and fruit, which is partly related to their different polyphenolic contents. The order of reducing power in E. ulmoides extracts at the amount of 2 mg/mL, compared with positive controls, was BHT > ascorbic acid > leaf> male flower > cortex > fruit. This order was similar to that of free radical scavenging effect but different from the metal chelating effect.

Antioxidant Activity in Linoleic Acid Emulsion

For evaluation of the antioxidant activity of E. ulmoides, the inhibition effect on the peroxidation of linoleic acid was investigated. As shown in Table 1, leaf extract could inhibit 59 percent peroxidation of linoleic acid; however, male flower extract, cortex extract and ascorbic acid only could suppress 40 percent, 29 percent and 30 percent of linoleic acid lipid peroxidation, respectively. Moreover, fruit extract showed very poor capacity to inhibit peroxidation of linoleic acid. The antioxidant activity of BHT was significantly higher than that any of all the plant extracts. The decreasing order of antioxidant activity in linoleic acid emulsion was BHT > leaf> male flower > ascorbic acid > cortex > fruit. This order was also close to that of free radical scavenging effect and reducing power, but different from that of metal chelating effect.

Total Polyphenolic Compounds

Polyphenolic compounds and flavonoids are the most active antioxidants derived from plants (Aruoma 1997). The antioxidant activity of water extract from E. ulmoides leaves and cortex was correlated to their polyphenol content (gallic acid equivalent, GAE) (Yen and Hsieh 1998), especially to their protocatechuic acid content (Yen and Hsieh 2000). To explain whether the polyphenolic compounds were the main antioxidant compounds in extracts of male flower and fruit, the total polyphenolics compounds content (gallic acid equivalent, GAE) of all the four extracts was determined by using the Folin-Ciocalteu method. As shown in Table 1, leaf extract contained much more polyphenolic compounds than other extracts, and extracts of male flower and cortex contained a similar number of polyphenolics. The antioxidant activity of the fruit extract was significantly lower than the other three extracts except for the metal chelating effect. Moreover, its polyphenolic compounds content was also significant less than other extracts. Although the polyphenolics content of the extract of male flower and cortex was similar, the antioxidant activity of male flower extract was significantly higher than that of cortex extract except for the metal ion chelating effect. From this it could be assumed that not only the content but also properties of polyphenolic compounds contribute to different activities in different extracts.

The above results indicate that the water extract from the leaf of E. ulmoides shows a stronger antioxidant activity than those from the other plant parts. It seems the reason for this difference just is that the polyphenolic content in the water extract from the leaf is much higher than in those from other parts. On the other hand, although there was no significant difference in the content of polyphenolics, the water extract from the male flower also showed significantly higher reducing power, DPPH scavenging effect and lipid peroxidation of inhibition than those from the cortex. This phenomenon suggests that the properties of polyphenolic compounds in the male flower and cortex were different in their chemical composition and structure. It also could be assumed from the results that not only the content but also properties of polyphenolic compounds contribute to different activities in different extracts. A further study, therefore, is necessary to discover the bioactive compounds in the male flower of E. ulmoides, which is currently under investigation in our laboratory.