Protective Effect of Flavonoids on Lymphocyte DNA Damage Using Comet Assay

Park, Yoo Kyoung; Jeon, Eun Jae; Kang, Myung Hee

Abstract

The present study was attempted to investigate and compare the antioxidant potency of several well-know flavonoids, antioxidant vitamin and commercially available popular beverages. The antioxidant potency was assessed by the effect on reducing oxidative DNA damage of human lymphocytes. Cellular oxidative DNA damage was measured by SCGE (single-cell gel electrophoresis), also known as comet assay. Lymphocytes were pre-treated for 30 minutes with wide ranges of doses of apigenin, kaempferol, luteolin, myricetin, rutin, quercetin, alpha-tocopherol (10,25,50,100,200,500,1000 micrometer) ,green tea extract or grape juice (10,50,100,250,500,1000 microgram/mL) followed by a H2O2(100 micrometer) treatment for 5 min as an oxidative stimulus. The physiological function of each antioxidant substance on oxidative DNA damage was analyzed as tail moment (tail length X percentage migrated DNA in tail) and expressed as relative DNA damage score after adjusting by the level of control treatment. Cells treated with H2O2 alone (positive control) had an extensive DNA damage compared with cells treated with phosphate buffered saline (PBS, negative control) or pre-treated with all the tested samples. Of all the six flavonoids, quercetin was the most potent antioxidant showing the lowest ED50 of 8.5 microgram/mL (concentration to produce 50% protection of relative DNA damage). The antoxidant potency of individual flavonoids were ranked as follows in a decreasing order; luteolin (18.4 microgram/mL), myricetin (19.0 microgram/mL) , rutin (22.2 microgram/mL) , apigenin (24,3 microgram/mL) , kaempferol (25.5 microgram/mL). The protective effect of alpha-tocopherol was substantially lower (highest ED50 value of 55.0 microgram/mL) than all the other flavonoids, while the protective effect was highest in green tea and grape juice with low ED5O value of 7.6 and 5.3, respectively. These results suggest that flavonoids, especially quercetin, and natural compounds from food product, green tea and grape juice, produced powerful anti-oxidative activities, even stronger than alpha-tocopherol. Taken together, supplementation of antioxidants to lymphocytes followed by oxidative stimulus inhibited damage to cellular DNA, supporting a protective effect against oxidative damage induced by reactive oxygen species.