CANCER PREVENTION RESEARCH □ ORIGINAL ARTICLE □ Screening of Antioxidant and Anticancer Effects from Flavonoids Kyoung Ah Kang1, Rui Zhang1, Mei Jing Piao1, Soyoon Park2, Jinny Park3, Ju Sun Kim4, Sam Sik Kang4 and Jin Won Hyun1 Departments of 1Biochemistry, 2Pathology, 3Medicine, College of Medicine, Cheju National University, Jeju 690-756, 4Natural Products Research Institute and College of Pharmacy, Seoul National University, Seoul 110-744, Korea Reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion, and hydroxyl radical lead to damages of cellular molecules and are a cause of cancer. To find the anticancer compounds that scavenge the ROS, we have screened the antioxidant effect against DPPH radical and H2O2 induced oxidative stress from 39 flavonoids at 10μg/ml. (+)-Catechin and epicatechin at 10μg/ml were found to scavenge DPPH radical and intracellular ROS. We also investigated whether two compounds may show anticancer effect against cancer cells by MTT reduction assay. As a result, epicatechin showed the strongest cytotoxicity to U937 cells among NCI-H460, HeLa, U937, and MCF-7 cells. (Cancer Prev Res 11, 235-239, 2006) ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Key Words: Oxidative stress, Reactive oxygen species, Anticancer flavonoids have been described.9) INTRODUCTION In present study, we have screened the antioxidant effect from flavonoids against radicals. In addition, from the selected Cancer is the leading cause of death in Korea. Among the two antioxidative flavonoids, it was investigated whether it may possible causes of cancer, damage to DNA and other cellular show anticancer effect against various cancer cells. molecules by reactive oxygen species (ROS) ranks high as a major culprit in the onset and development of cancer.1∼3) MATERIALS AND METHODS Oxidative stress induces gene mutation and promotes carcino- 1. Flavonoids genesis, thereby leading to cancer.4,5) Experimental studies support that dietary antioxidants (eg, vitamin E, vitamin C, β- Flavonoid compounds were obtained from Dr. Sam Sik Kang carotene, and other phytochemicals) as well as endogenous (Seoul National University, Seoul, Korea). antioxidants (eg, glutathione) that neutralize or trap ROS act 2. Reagents as cancer preventive agents.3,6) Dietary flavonoids are natural antioxidants7) and found in various plant products such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2',7'-dich- fruits, leaves, seeds, oils and plant-derived beverages such as lorodihydrofluorescein diacetate (DCF-DA), and [3-(4,5-dime- tea and wine.8) Humans have consumed flavonoids and other thylthiazol-2-yl)-2,5-diphenyltetrazolium] bromide (MTT) were dietary phenolics compounds and over 4000 different naturally purchased from Sigma Chemical Company (St. Louis, MO, 책임저자：현진원, ꂕ 690-756, 제주도 제주시 제주대학로 66 Correspondence to：Jin Won Hyun 제주대학교 의과대학 생화학교실 Department of Biochemistry, College of Medicine, Cheju National Tel: 064-754-3838, Fax: 064-726-4152 University, 66, Jejudaehak-no, Jeju 690-756, Korea E-mail: [email protected] Tel:+82-64-754-3838, Fax:+82-64-726-4152 접수일：2006년 8월 24일, 게재승인일：2006년 9월 20일 E-mail: [email protected] 235 236 Cancer Prevention Research Vol. 11, No. 3, 2006 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ USA). After 1 h, the amount of residual DPPH was determined at 520 nm using a spectrophotometer.10) 3. Cell culture To study the antioxidative effects of flavonoids against DPPH radical scavenging activity (%) = oxidative stress, it was used V79-4 (Chinese hamster lung OD of DPPH radical treatment- (OD of Flavonoids+ DPPH radical treatment) fibroblasts cells). To study the cytotoxic effect of flavonoids ×100 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ against cancer cells, NCI-H460 (human non-small cell lung), OD of DPPH radical treatment HeLa (human cervix cancer cell), U937 (human monocytic leukemia cell) and MCF-7 (human breast cancer cell) from the 5. Intracellular ROS measurement American Type Culture Collection were used. These cancer cells were maintained at 37oC in an incubator with a The DCF-DA method was used to detect the intracellular 11) humidified atmosphere of 5% CO2 and were cultured in ROS level. DCF-DA diffuses into cells, where it is hydrolyzed Dulbecco's modified Eagle's medium and RPMI 1640 medium by intracellular esterase to polar 2',7'-dichlorodihydrofluo- containing 10% heat-inactivated fetal calf serum, streptomycin rescein. This non-fluorescent fluorescein analog gets trapped (100μg/ml) and penicillin (100 units/ml). inside the cells and is oxidized by intracellular oxidants to a highly fluorescent, 2',7'-dichlorofluorescein. The V79-4 cells 4. DPPH radical scavenging activity were seeded in a 96 well plate. Sixteen hours after plating, the Ten μg/ml of flavonoids were added to a 1×10-4 M cells were treated with flavonoids at 10μg/ml and 1 h later, solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in 1 mM H2O2 was added to the plate. The cells were incubated methanol, and the reaction mixture was shaken vigorously. for an additional 30 min at 37oC. After addition of 25μM of Table 1. Effect of flavonoids on scavenging DPPH ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Flavonoids 10μg/ml Flavonoids 10μg/ml ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Acacetin 2.7±0.4% Kenusanone 9.0±0.9% Apigenin 1.7±0.3% Linarin 2.9±0.5% Apigenin 7-O-neohesperidoside 1.7±0.4% Liquiritigenin 1.5±0.1% Astragalin 2.1±0.5% Maackiain 1.5±0.6% Baicalin 6.6±1.7% Naringenin 1.1±0.1% (+)-Catechin 29.1±1.2%* Naringin 4.3±0.7% Chrysin 2.4±0.8% Poncirin 5.2±0.8% Diosmetin 7-O-glucoside 2.4±1.0% Quercetin 9.5±1.5% Echinoisoflavanone 1.2±0.4% Quercetin coumaroyl glucorhamnoside 3.4±0.3% Echinoisosohoranone 3.1±0.9% Quercetin 3-O-2",6"- 4.1±0.4% Epicatechin 35.5±2.3%* dirhamnosylglucoside Eupatilin 1.1±0.1% Robinin 1.8±0.2% Genistein 5.5±0.3% Rutin 9.4±1.2% Genistin 1.9±0.1% Sophoflavescenol 1.9±0.2% Hesperidin 3.9±0.1% Sophoraflavanone D 10.1±1.0% Icariin 1.3±0.7% Sophoraflavanone G 1.1±0.1% Isoliquiritigenin 6.6±1.2% Spinosin 2.7±0.3% Isoquercitrin 12.1±1.1% Trifolirhizin 1.6±0.2% Isorhamnetin 1.6±0.6% Vitexicarpin 1.3±0.4% Isoxanthohumol 2.1±0.4% Vitexin 1.4±0.2% ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ The amount of DPPH radicals was determined spectrophotometrically. The measurement was made in triplicate and values are expressed as mean±standard error. *Significantly different from control (p＜0.05). Kyoung Ah Kang, et al：Screening of Antioxidant and Anticancer Effects from Flavonoids 237 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ DCF-DA solution, the fluorescence of 2',7'-dichlorofluorescein Cell viability (%) = was detected at 485 nm excitation and at 535 nm emission OD of Control-OD of Flavonoids treatment ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ ×100 using a PerkinElmer LS-5B spectrofluorometer. OD of Control Intracellular ROS scavenging activity (%) = 7. Statistical analysis OD of H2O2 treatment- (OD of Flavonoids+H O treatment) 2 2 ×100 All the measurements were made in triplicate and all values ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ OD of H2O2 treatment were represented as means±standard error. The results were subjected to an analysis of the variance (ANOVA) using the Tukey test to analyze the difference. p＜0.05 were considered 6. Cell viability significantly. The cell viability of flavonoids against various cancer cells are determined using the [3-(4,5-dimethylthiazol-2-yl)-2,5- RESULTS AND DISCUSSION diphenyltetrazolium] bromide (MTT) assay, which is based on the reduction of a tetrazolium salt by mitochondrial dehy- To find compounds as the antioxidant from flavonoids, 39 drogenase in the viable cells.12) Flavonoids were treated and flavonoids at 10μg/ml were tested by measuring their incubated for 24 h. The MTT solution was added and scavenging effects of DPPH radical and intracellular ROS. As incubated for 4 h. The formazan crystals in each well were shown in Tables 1 and 2, (+)-catechin and epicatechin showed dissolved in 150 ul dimethylsulfoxide and the absorbance was radical scavenging effect in the following two assays. In DPPH measured at 540 nm. radical scavenging effect, (+)-catechin and epicatechin showed Table 2. Effect of flavonoids on scavenging intracellular ROS induced by H2O 2 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Flavonoids 10μg/ml Flavonoids 10μg/ml ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Acacetin 3.5±0.4% Kenusanone 35.3±3.2% Apigenin 3.2±0.2% Linarin 16.9±1.9% Apigenin 7-O-neohesperidoside 10.2±1.0% Liquiritigenin 8.2±1.0% Astragalin 17.6±4.2% Maackiain 8.5±0.9% Baicalin 57.7±1.3% Naringenin 20.2±0.6% (+)-Catechin 85.1±0.7%* Naringin 38.7±1.9% Chrysin 16.3±3.0% Poncirin 25.6±1.2% Diosmetin 7-O-glucoside 6.1±0.2% Quercetin 78.5±1.7% Echinoisoflavanone 5.2±0.3% Quercetin coumaroyl glucorhamnoside 73.4±2.0% Echinoisosohoranone 4.1±0.7% Quercetin 3-O-2",6"-dirhamnosylglucoside 58.0±1.6% Epicatechin 83.3±1.5%* Robinin 31.9±1.5% Eupatilin 8.2±0.3% Rutin 72.9±1.9% Genistein 15.6±1.3% Sophoflavescenol 35.3±2.1% Genistin