Anti-Oxidative and Anti-Proliferative Activity on Human Prostate Cancer Cells Lines of the Phenolic Compounds from Corylopsis coreana Uyeki

Fifteen phenolic compounds, including three caffeoyl derivatives, four gallotannins, three ellagitannins and five flavonoids, were isolated from an 80% MeOH extract of the leaves of Corylopsis coreana Uyeki (Korean winter hazel; CL). The anti-oxidative activities [1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and xanthine oxidase superoxide scavenging activities (NBT)] and the anti-proliferative activity on human prostate cancer cell lines (DU145 and LNCaP) were also evaluated.

activities against various forms of cancer were also reported [5]. This paper describes the isolation of compounds from the leaves of CL and evaluation of its anti-oxidative activities and the anti-proliferation properties of the isolated compounds.

Cell Viability and Inhibition of Cancer Cell Proliferation
The cell viability was measured using the MTT assay ( Figure 2), which is based on the mitochondria-dependent reduction of MTT to formazan [24]. In order to evaluate the anti-proliferation activities of the compounds 1-15 from CL, cell viability were tested on DU145 and LNCaP prostate cancer cells. The anti-proliferative effects of hydrolysable tannins in sarcoma cells and HeLa cells were reported [25] and the functional groups the hydrolysable tannins are also important factors determining their anti-proliferation activity [26]. Among the ellagitannins 8-10, compound 10 showed higher androgen sensitive anti-proliferation activity, suggesting the importance of the HHDP group. Since 9 was more potent than 10, the presence of both HHDP and galloyl groups might be necessary. Compound 9 was also more potent than 8, suggesting the importance of a galloyl group in the C-l position [27]. The compounds 8, 9 and 10 inhibited the proliferation of both DU145 and LNCaP prostate cancer cells (Table 3).
43.08 ± 0.63 a >100 a 10 40 Values represent means ± S.D. of three determinations. Values bearing different superscripts in the same column are significantly different (p < 0.05).

Plant Material
The leaves of CL (1.8 kg) were collected from the Korea Forest Research Institute, Suwon, Korea in September 2010 and certified by Minwon Lee (Phamacognosy Lab, College of Pharmacy, Chung-Ang University). The voucher specimen (CL2010-9) was deposited at the herbarium of the College of Pharmacy, Chung-Ang University.

Anti-Proliferation and Cytotoxicity Assays
3.5.1. Cell Culture RAW 264.7 macrophage, DU-145 and LNCaP human prostate cancer cell lines were purchased from the Korean Cell Line Bank. The RAW 264.7 macrophage cell and human prostate cancer cell lines was grown at 37 °C in a humidified atmosphere (5% CO 2 ) in DMEM (Sigma) and RPMI (Sigma) containing 10% fetal bovine serum, 10 IU/mL penicillin G and 100 μg/mL streptomycin (Gibco BRL, Grand Island, NY, USA).

Measurement of Cell Viability
After culturing of RAW 264.7 macrophage (3.5 × 10 5 cells/200 μL medium) in 96-well plates and incubating for 2 h, in 96-well plates and incubating for 24 h, the cells were treated with the test samples. The cells were incubated for an additional 24 h, and the medium was replaced with fresh medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma). Incubation was continued for 4 h at 37 °C. The medium was then removed and the MTT-formazan produced was dissolved in dimethyl sulfoxide (DMSO). The extent of the reduction of MTT to dark purple crystals within the cells was quantified by measuring the absorbance at 540 nm using the microplate reader (Tecan) [24].

Anti-Proliferation Assays
After culturing of DU145 and LNCaP human prostate cancer cell lines (1 × 10 4 cells/ 200 μL medium) in 24-well plates and incubating for 24 h, the cells were treated with the test samples. Plates were incubated for 72 h after which bioassays were performed. LNCaP cells were handled in a similar manner, and the medium was replaced with fresh medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma). Incubation was continued for 4 h at 37 °C. The medium was then removed and the MTT-formazan produced was dissolved in dimethyl sulfoxide (DMSO). The extent of the reduction of MTT to dark purple crystals within the cells was quantified by measuring the absorbance at 540 nm using the microplate reader (Tecan) [24].

Statistical Analysis
All data are expressed as mean ± S.D. Values were performed by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls (S-N-K) test using the SPSS software package; the values were considered significantly different when the p value was less than 0.05.

Conclusions
Fifteen phenolic compounds 1-15, inclusing three caffeoyl derivatives 1-3, four gallotannins 4-7, three ellagitannins 8-10 and five flavonoids 11-15 were isolated from CL. DPPH radical scavenging and superoxide scavenging activity, as well as, anti-proliferative activity on prostate cancer call lines (DU145 and LNCaP) of the isolated compounds were evaluated. As a results, the ellagitannin derivatives 8-10 showed potent anti-oxidative and anti-proliferative activities on prostate cancer cell lines. These results suggested that CL extract and its phenolic compounds could potentially be developed as ingredients with anti-oxidative and androgen sensitive anti-proliferation activity.