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Commercial bark of stem powder of Ulmus davidiana (U. davidiana) var. japonica, which was dried under room temperature was purchased. Ultra-fine U. davidiana powder (UFP) was manufactured by the dry milling in Apexcel Co. (Pohang, Korea) and stored at room temperature. The average particle size of UFP was in the range of 1-3 μm and commercial powder (CP) was about 100 μm. All chemicals and reagents were purchased from Sigma Chemicals Co. (St. Louis, MO, USA).

For ethanol (EtOH) extraction, three hundred grams of either CP or UFP were dissolved in 1,000 mL of EtOH after 24 hr soaking. Then, soluble collected EtOH fraction was concentrated to about 20 mL volume using a vacuum evaporator and kept in a refrigerator until use. For hot-water extraction, 300 g of either CP or UFP U. davidiana var. japonica were boiled in 1,000 mL for 2 hr, filtered and concentrated with freeze-dryer.

The antioxidant activity was determined with its ability in stabilizing the free radical DPPH (1,1-diphenyl-2-picrylhydrazyl). The mixture containing 40 μL of extracts (4 mg/mL dissolved in dimethyl sulfoxide (DMSO)) and 760 μL of 300 μM DPPH solution was heated at 37℃ for 30 min and the absorbance was determined at 515 nm. The DPPH ability was calculated and all determination was triplicated.

DPPH radical scavenging activity (%) = [(A₀-A₁)/A₀]x100,

where A₀ was the absorbance of the control and A₁ was the absorbance of the sample.

ABTS (2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenger ability was measured with modification (Re et al. 1999). Seven mM of ABTS reagent was mixed with 2.45 mM potassium persulfate and stood for 12-16 hr in dark. After ABTS radical was produced, it was diluted upto 0.70 absorbance value at 734 nm. The 40 uL of sample (4 mg/mL dissolved in DMSO) and 760 uL ABTS radical solution were mixed and incubated at room temperature for 10 min. At 734 nm, the absorbance was measured and the ABTS ability was calculated. All measurements were in triplicate.

ABTS radical scavenging activity (%) = [(A₀-A₁)/A₀]x100,

where A₀ was the absorbance of the control and A₁ was the absorbance of sample.

Hydroxyl radical scavenger ability was measured with a few modification (Smirnoff & Cumbes 1989). The mixture contained 250 μL of FeSO₄ (1.5 mM), 175 μL of hydrogen peroxide (6 mM) and 300 μL of sodium salicylate (20 mM) and reacted for 30 min at 37℃. The absorbance was determined at 562 nm and activity was calculated. All determination was triplicated.

The reducing power was examined by the method of Oyaizu(1986). Different concentrations of 2.5 mL of each samples were reacted with 2.5 mL (200 mM) of sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. After 20 min incubation at 50℃, 2.5 mL of 10% trichloroacetic acid (w/v) were added. Then, the mixture was centrifuged at 650 rpm for 10 min, and the upper layer (5 mL) was collected. Five mL of deionized water and 1 mL of 0.1% of ferric chloride were added and the absorbance was measured at 700 nm.

One mg of the sample was mixed with 2 mL of Foiln-Denis reagent and 2 mL of 35% sodium carbonate, mixed vigorously and filled up to 10 mL with distilled water. After 30 min incubation at ambient temperature, the absorbance was measured at 765 nm, The contents of phenolic compound were determined through a standard curve prepared at different concentrations of tannic acid.

Statistical analysis system (version 9.0, SAS Institute Inc. Cary, NC, USA) was used. ANOVA was used to test significant differences among the samples, and means were performed by Duncan’s multiple range test (p<0.05).

The total phenolic contents of either CP or UFP U. davidiana ver. japonica were shown in Table 1. The phenolic content of CP and UFP extraction samples in EtOH were 40.38 and 65.61 mg tannic acid equivalent/g, respectively. However, a lower content of phenolic compound was found in hotwater such as 39.68 mg tannic acid equivalent/g in CP and 45.62 mg tannic acid equivalent/g in UFP U. davidiana var. japonica.

Earlier study showed that phenolic compounds have been shown to possess a high antioxidant property (Guo & Wang 2007). Other previous study have also shown a colse relationship between total phenolic content and an antioxidant capacity in some berry crops (Kalt et al. 1999).

Polyphenols belong to a several kinds of materials with a high antioxidant actions. Those compounds are generally known to stabilize oxygen-derived free radicals by contributing a hydrogen atom to the free radical (Guo & Wang 2007). In searching for sources of natural antioxidants, the radical scavenging activities of medicinal plants and fruits have been studied in the last few decades (Guo & Wang 2007).

The antioxidant activity of DPPH is thought to be due to their hydrogen-donating ability. In this study, scavenging activity of EtOH or hot-water extract on DPPH was compared between CP and UFP U. davidiana var. japonica and shown in Fig. 1.

Fig. 1. 
DPPH radical scavenging activities of commercial or ultra-fine Ulmus davidiana powder at different concentrations

DPPH radical scavenging activity increased with increasing extract concentrations (Fig. 1). At every concentrations, UFP-EtOH showed the highest activity among other samples. Also, the DPPH scavenging activity was significantly higher at lower concentrations (0.32, 1.6, and 8 μg/mL) (Fig. 1). The activity reached up to 81.8 and 76.7% at 200 μg/mL concentration in UFP-water and CP-water, which were significantly lower activities compared with those in EtOH extracts (90.4 and 91.8%). DPPH radical scavenging activity of water extract was decreased dramatically at 40 μg/mL concentration.

Both CP-EtOH and UFP-EtOH showed a strong antioxidant activity (IC₅₀ at 12.56 and 6.75 μg/mL), whereas IC₅₀ which reflects 50% depletion of DPPH free radicals in CP-water and UFP-water were 70.05 and 31.64 μg/mL (data not shown). These data were similar to those of other study, which evaluated the free radical scavenging activity of U. davidiana extracts with various solvents (Guo & Wang 2007). A high positive correlation has been found between total polyphenolic contents and DPPH radical scavenging ability (Guo & Wang 2007).

Therefore, the present study indicated that EtOH extract showed the highest antioxidative activity compared with other samples, therefore, EtOH extract may act as free radical inhibitors or scavengers.

Scavenging activity of EtOH or hot-water extracts on ABTS was compared between CP and UFP U. davidiana var. japonica and shown in Fig. 2. There was no difference in samples at 40 and 200 μg/mL concentrations (p<0.05). At 200 μg/mL, scavenging activities were found in the range of 91.6 to 98.2%, and over 89% of scavenging activity was shown in all samples at 40 μg/mL concentration. The IC₅₀ values was 3.76 and 2.96 μg/mL of CP-EtOH and UFP-EtOH (data not shown) and UFP and CP in hot-water extraction showed the similar scavenging activity with IC₅₀ (8.62 and 9.48 μg/mL, respectively). However, extracts of hotwater showed the significantly lower activity than that of EtOH at 8 μg/mL and lower concentrations (p<0.05). These results indicated that both UFP and CP extracts of U. davidiana var. japonica showed a strong antioxidative activity on ABTS at high concentration, regardless of EtOH and hot-water extraction.

Fig. 2. 
ABTS radical scavenging activities of commercial or ultra-fine Ulmus davidiana powder at different concentrations.

In hydroxyl radical scavenging assay, both activities of CP and UFP in EtOH extraction increased moderately up to 73.5 and 67.9% at 200 μg/mL (Fig. 3). There was no dramatic difference between samples. Hot-water extracts showed slightly lower activities at 40 and 200 μg/ml concentration than EtOH extracts. Also, not much difference was found between CP and UFP in hydroxyl scavenging activity in both EtOH and hot-water extraction.

Fig. 3. 
Hydroxyl radical scavenging activities of commercial or ultra-fine Ulmus davidiana powder at different concentrations

Studies of effective hydroxyl radical scavengers indicate that the activity is closely related to the phenolic contents (Guo & Wang 2007). Phenolic compounds are widely contained in both edible and inedible plants, and those have been known to show various functional effects (Guo & Wang 2007). These antioxidant activity is mostly due to their redox properties, which can be an aspects in absorbing free radicals (Siriwardhana et al. 2003). Therefore, a slightly higher activity found in EtOH extracts of UFP is likely due to higher contents of phenolic compound in samples.

The reducing power may be a significant indicator of its potential antioxidant ability (Jung et al. 2008). Fig. 4 presents the reductive capabilities of both CP and UFP U. davidiana var. japonica on EtOH and hot-water. The significantly high reducing power was shown in UFP-EtOH group, compared with others at every concentrations except for 0.32 μg/mL (p<0.05).

Fig. 4. 
Reducing power of commercial Ulmus davidiana powder at different concentrations

Reducing power is closely related to the reductones, which have been known as an antioxidant agent by giving a hydrogen atom (Duh 1998). In our study, EtOH extracts of UFP showed a higher reducing activity, which suggests that there may be some reductones contained in UFP U. davidiana var. japonica.

In present, BHA and BHT, the synthetic antioxidants, are commomly used to reduce lipid peroxidation and to prolong the shelf-life of food products. Recent studies have been focused on natural compounds as a natural antioxidants derived from medicinal plants. In consequence, it is widely accepted that some of synthetic antioxidants need to be replaced because of their potential health risk (Li et al. 2008).

Several previous studies have indicated that U. davidiana extracts of various solvents showed a strong antioxidant activity (Guo & Wang 2007; Jung et al. 2008; Ahn & Park 2010; Park 2011). However, no study has been done to evaluate the effect of ultra-fine size for extraction process. The results of the various inhibitor assays revealed that EtOH extract of U. davidiana var. japonica could be considered as an effective additive in food and pharmaceutical agent.