The Olea europaea olive leaf was harvested from 2011.7 to 2012.6, dried at room temperature, then grinded into powder. For extraction, two solvents (methanol and water) were used to fractionate the soluble compounds from the O. europaea L. leaves. The fresh leaf powder (50 g per month) was mixed with water (1:20, w/v) at 50˚C for 3 days and the extraction was repeated three times. Using solvent methanol, the extraction was carried out at room temperature for 3 days and repeated three times, the solvents from the combined extracts were evaporated by vacuum rotary evaporator. After filtration, 1 g of extracts obtained were then transferred to vials and kept in the dark at −20˚C prior to use. The rest part of extracts was combined and dissolved in water, filtered through Whatman filter paper, concentrated with a vacuum evaporator and the water-soluble part was stored for next chromatography use.

10 g Water-soluble fraction was absorbed by D101 column, eluted with water and methanol respectively, named C1 and C2 fraction. The methanol part C2, was recovered with a vacuum evaporator, then C2 fraction was separated by Sephadex LH-20, gradient elution with 250 mL of water-methanol system (0%, 5%, 10%, 20%, 40%, 60%, 100%) respectively. By TLC detection, the elution was divided into eight fractions, named C3 - C10 and stored for next use.

The preparation of tyrosinase was described as following: 100 g fresh tomato was purchased from local market. After decorticated and chipped, the tomato was soaked in 200 mL phosphate buffer solution (0.2 M, pH 7.2), grinded into power and filtered by paper, the solution was centrifuged at 4000 g × 5 min. The supernatant was suction and stored at −4˚C for next use.

The determination of tyrosinase was performed used LDOPA as substrate according to the method described before [7] with slight modification. tyrosinase were determined by spectrophotometrically measuring the rate of dopachrome formation at 475 nm (ε = 3700 M⁻¹·cm⁻¹) by using a UV-1800 spectrophotometer. One unit of enzymatic activity was defined as the amount of enzyme transforming 1 μmol of L-tyrosine or L-DOPA/min. First, 0.8 mL of a 2.5 mM L-DOPA was mixed with 2.4 mL of 0.067 M phosphate buffer solution (PBS, pH 6.6) and incubated at 37˚C for 10 min. Then 0.8 mL tyrosinase solution was added to the mixture. This solution was immediately monitored for the formation of dopachrome by measuring the linear increase in optical density at 475 nm for 5 min. Triplicate measurements were recorded. The activity of L-tyrosinase activity was calculated by formula:

Tyrosinase activity = k/(10⁻⁶ ε × V × 2.5 × 0.1), in which k is slope of curve, ε = 3700 (mol/L)⁻¹, V is the test volume of enzyme (here is 0.8 mL).

The determination of tyrosinase was performed usin LDOPA as substrate according to the method described before [7-10] with slight modification. First, 0.8 mL of a 2.5 mM L-DOPA was mixed with 2.4 mL of 0.067 M PBS (pH 6.6) and incubated at 37˚C for 10 min. Then, 0.8 mL of 2 mg/mL samples and 0.8 mL tyrosinase solution were added in this order to the mixture. This solution was immediately monitored for the formation of dopachrome by measuring the linear increase in optical density at 475 nm for 5 min. Triplicate measurements were recorded. The inhibition of tyrosinase activity was calculated with the following formula:

.

Where A was with sample and L-DOPA, B was with sample without L-DOPA, C was with substrate without sample, D was without substrate and samples.

To study in depth tyrosinase inhibition by OLE, we studied OLE effect on tyrosinase inhibition type, and the kinetic parameters (K_I).

For determination of inhibition type, various concentrations of L-DOPA (0.0625 - 10 mM) as the substrate were used. Inhibitory kinetics of samples was analyzed by Lineweaver-Burk plots. The kinetic data were plotted as 1/activity (1/V) versus 1/substrate concentration (1/S), according to the method of Lineweaver-Burk.

The scavenging activity of the extract on 1,1-diphenyl- 2-picrylhydrazyl (DPPH), was determined according to the method of Tevfik Ozen [11] with slight modification. 0.1 mL of samples (1 mg/mL) or standard antioxidants solution (vitamin C, 100 - 6.25 μg/mL) was added to a methanolic solution (0.9 mL, 1.5 × 10⁻⁴ M) of DPPH radical. The mixture was shaken vigorously and kept in the dark at room temperature for 30 mins, the absorbance of the resulting solution was then measured spectrophotometrically at 517 nm. The reduction in the absorbance of the DPPH solution indicated the free radical scavenging activities of those antioxidants. The DPPH radicalscavenging activity was calculated according to the following formula:

Scavenging rate E (%) = (1 −A)/A₀) ×100 .

Where A₀ is the absorbance of the control reaction (containing all reagents except the tested compound), and A is the absorbance of the tested compound.

Superoxide radicals were generated by pyrogallic acid method with some modification [12-14]. The reaction mixture contained 2 mL sample (0.0 - 1 mg/mL) dissolved in 2.25 mL Tris-HCl buffer (0.05 M, pH 8.0). After incubated at 25˚C for 10 mins, the reaction was initiated by adding 0.075 mL of pyrogallol solution (10 mM) for 4 mins, finally 0.02 mL of thick hydrochloric acid was added for termination the reaction. The optical density (OD) of the resulting solution was measured in triplicate at 420 nm using a spectrophotometer. Ascorbic acid was used as positive control. In blank, sample solution was substituted with Tris-HCl buffer.

The inhibition percentage of superoxide anion generation was calculated using the following formula equation:

.

Where A was the absorbance of the test sample, and A₀ was absorbance of the blank. Vc was set as positive control.

The reducing power of extracts was determined according to the Method described before [11,13,15,16] with slight modification. A 1 mL of extract sample or standard antioxidant (ascorbic acid) was mixed with 2.5 mL of 0.2 M phosphate buffer saline (pH 6.6) and 2.5 mL of 1% (w/v) potassium ferricyanide [K₃Fe(CN)₆]. After the mixture was incubated at 50˚C for 20 min, 2.5 mL of 10% (w/v) trichloroacetic acid was added, and the mixture was centrifuged at 1400 ×g for 10 min. The upper layer of 2.5 mL solution was mixed with 2.5 mL deionized water and 0.5 mL of 0.1% (w/v) FeCl₃ for 10 min, and then the absorbance was measured at 700 nm. Higher absorbance of the reaction mixture indicated greater reductive potential.

Samples were prepared as Figure 1. In brief, OLE was collected among 12 month and treated by chromatogramphy, finally OLE was divided into 10 fractions by its polarity.

As shown in Figure 2, there is little difference in the extracing solvent. Reducing power and DPPH scavenging showed more active than inhibition of Superoxide anion radical.

Tyrosinase inhibitory activity was listed as Figure 3. Among 12 months, the leaf collected from autumn showed the highest acitivity, it give us a tip that autumn maybe is the better time to collect the samples.