The Antioxidation and Antiproliferation Activity of New Flavonoids from the Leaves and Stems of Cinnamomum reticulatum Hayate

The Cinnamomum species have used in folk medicine. Cinnamomum species have many active chemical compounds with anti-oxidation, anti-cancer, anti-infection and anti-inflammatory activities [1,2]. Cinnamoum reticulatum Hayata (Lauraceae) is an evergreen tree, a tree native to Taiwan. Previous studies, we have isolated compounds including reticuol, isoreticulide, 4-hydrox-3methoxyphenethyl derivate, reticumanone, reticuone, cinnaretamine and isoobtusilactone A from the stems and leaves of Cinnamomum reticulatum [3-5].


Cell culture
Human lung cancer and human breast cancer cell lines A549, NCI-H460 MCF-7 MDA-MB231 were obtained from BCRC (Bioresoure Collection and Research Center, Hsinchu, Taiwan). They were cultured in MEM medium supplemented with 10% FBS, 10 μg/mL of penicillin, 10 μg/mL of streptomycin and 0.25 μg/mL of amphotericin B. The cells were maintained in a humidified incubator at 37 °C in 5% CO 2 .

Biological activity
IC 50 is a measure of the effectiveness of a drug in inhibiting biological or biochemical function. In the present study, the antiproliferation activity of the two compounds was determined by XTT assay and the results were presented in Table 1. Based on the IC 50 value of two compounds, kaempferol-3-O-(3",4"-di-E-p-coumaroyl)α-L-rhamnopyranoside (2) has better anti-proliferation activity than kaempferol-3-O-(2",4"-di-E-p-coumaroyl)-α-L-rhamnopyranoside (1) in cancer cells.

Cell viability assay-XTT assay
The XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-carboxanilide) assay was used to determine cell viability and proliferation. The cell lines were seeded in 96-well culture plates (1×10 4 cells/well). XTT were obtained from Sigma-Aldrich (GmbH, Sternheim, Germany). After seeding cells for 24 h, various different concentrations of compounds were added. After the treatment, the medium was replaced with fresh medium without drugs. XTT reagent was added to each well and cultured for 3 hours. The optical density (OD) values of the supernatant were measured at 492 nm and 690 nm (reference wavelength). All experiments were repeated at least three times.

Determination of DPPH radical scavenging capacity
DPPH is an abbreviation for an organic chemical compound 2,2-diphenyl-1-picrylhydrazyl. It is a dark-colored crystalline powder composed of stable free-radical molecules. The mechanism of its radical scavenging activity is the antioxidant transfer of an electron or a hydrogen atom to DPPH. DPPH were obtained from Sigma-Aldrich (GmbH, Sternheim, Germany).The measurement of radical scavenging properties of compounds was carried out according to the method of Blois [9]. Various concentrations of the three compounds were added to 0.1 M of stable DPPH (60 μmole/L) solution. When DPPH reacts with hydrogen-donating antioxidant, it is reduced, resulting in a decrease in absorbance at 517 nm. The analyzed time interval was 10 min per point, up to 30 min by using UV-Vis spectrophotometer (Jasco V-530, Japan Servo Co.Ltd., and Tokyo, Japan). Vitamin C was used as a positive control. Measurements were taken in triplicate. The DPPH• radical scavenging activity (%) was determined as: 1 − [(A control − A sample )/A control ] × 100.

Reducing power
Briefly, various concentrations of test samples were mixed with 67 mM phosphate buffer (pH 6.8, 0.085 mL) and 20% potassium ferric cyanide [K 3 Fe(CN) 6 , 2.5 μL) The mixture was incubated at 50ºC for 20 min, and trichloroacetic acid (10%, 0.16 mL) was then added to the mixture that was then centrifuged for 10 min at 3000 g. The upper layer of the solution (75 μL) was mixed with 2% FeCl 3 (25 μL), and the absorbance was measured with a 96-well plate spectrophotometer at 700 nm. 3-tert-butyl-4-hydroxyanisole (BHA) was used as a positive control. A higher absorbance demonstrates a higher reductive capability.

Statistical analysis
All experiments were carried out at three times and at least triplicate. The results were expressed as the average of the mean values ± standard deviation (SD). Analysis of the data was done with SigmaPlot software (Version 8.0, SPSS Scientific, and Chicago, IL, USA) and SigmaStat (Version 2.03, SPSS Scientific) run on an IBM-compatible computer.

K a e m p f e r o l -3 -
Flavonoids possess anti-oxidative effects as free radical scavengers and metal ion cheaters properties associated with the phenolic hydroxyl groups attached to ring structures. Intensity of the antioxidant activity of a flavonoid strongly depends on its chemical structure. There is a great deal of discussion and contradiction regarding the structure antioxidant activity relationships of flavonoids. However, it is wellaccepted that the antioxidant activity of flavonoids is markedly influenced by the number and position of hydroxyl groups on the B and A rings, and by the extent of conjugation between the B and C rings. On the basis of many previous and recent findings, it seems that favorable general structural requirements for effective radical scavenging and/or the antioxidative potential of flavonoids follow the famous three Bors' criteria: a) The o-hydroxy structure in the B ring, which confers high stability to the flavonoid phenoxyl radicals via hydrogen bonding or by expanded electron delocalization; b) The C2-C3 double bond (in conjugation with the 4-oxo group), which determines the coplanarity of the heteroring and participates in radical stabilization via electron delocalization over all three ring systems; c) The presence of both 3-OH and 5-OH groups for the maximal radical scavenging capacity and the strongest radical absorption (i.e., 1 and 2).
Taken these results together, the two flavonoids possess antioxidation and cytotoxic activity in the cancer cells. In the future, the exact mechanisms of anti-cancer activity of the compounds can be further examined.