본 연구는 유근피(root bark of Ulmus davidiana)에서 분리한 화합물인 hederagenin 3-O-b-D-glucopyranosyl($1{ ightarrow}3$)-a-L-rhamnopyranosyl($1{ ightarrow}2$)-a-L-arabinopyranoside (HDL)을 이용하여 $CoCl_2$에 의해 생성된 ROS에 대한 항산화 기전을 밝히기 위하여, ROS 생성과 관련된 산화 효소 및 항산화 효소에 대한 저해효과를 조사하였다. 또한 HDL이 $CoCl_2$에 의해 생성된 ROS의 조절을 통해 산화적 스트레스와 관련된 단백질 발현 및 세포주기에 미치는 영향을 조사하였다. 그 결과 HDL은 $CoCl_2$에 의해 유발된 xanthine oxidase와 $H_2O_2$ 생성 증가를 억제하였고, 산화와 관련된 SOD, CAT의 활성을 증가시켜 $H_2O_2$의 가수분해를 촉진하였다. 그리고 HDL은 $CoCl_2$에 의해 유발된 ferritin의 손상과 ferritin iron의 방출을 억제하 였으며, 지질과산화의 증가를 억제하였다. 뿐만 아니라 HDL은 $CoCl_2$에 의해 증가된 G1 phase의 세포를 감소시켰으며, 세포주기와 관련된 p53 및 $p21^{CIP1/WAF1}$의 발현을 감소시켰다. 이러한 연구결과들은 HDL이 천연물로부터 유래한 독성이 없는 항산화제로서의 가능성을 제시한다.
We investigated the antioxidant effects of hederagenin 3-O-b-D-glucopyranosyl($1{ ightarrow}3$)-a-L-rhamnopyranosyl($1{ ightarrow}2$)-a-L-arabinopyranoside (HDL) isolated from root bark of Ulmus davidiana on the activity of enzymes related to reactive oxygen species (ROS) in human osteosarcoma U2OS cells. Cobalt chloride ($CoCl_2$), a transition metal, was used as an inducer of oxidative stress, generating hydrogen peroxide ($H_2O_2$) via increasing xanthine oxidase (XO) activity. The increased levels of $H_2O_2$, XO, ferritin, and ferritin iron by $CoCl_2$ were diminished effectively by co-treatment with HDL in U2OS cells. Furthermore, decreased levels of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) by $CoCl_2$ were highly increased by co-treatment with HDL in U2OS cells; however, the levels of glutathione peroxidase (GPx) did not change. The increased contents of TBARS related to lipid peroxidation were significantly reduced by HDL in U2OS cells. The concentration of GSH changed in a pattern that went against regulated TBARS by $CoCl_2$ and HDL. We examined the expression of p53, $p21^{CIP1/WAF1}$, and $p27^{KIP1}$ proteins related to oxidative stress and cell cycle regulation. As a result, the expression of $p27^{KIP1}$ modulated by $CoCl_2$ was not changed by HDL. However, the expression of p53 and $p21^{CIP1/WAF}$ increased by $CoCl_2$ was reduced by HDL in U2OS cells. Together with alteration of p53 and $p21^{CIP1/WAF1}$ proteins, the accumulated cells at G1 phase by $CoCl_2$ was decreased by HDL in U2OS cells. Our data suggests that HDL inhibits $CoCl_2$-generated ROS in U2OS cells, providing potentially new antioxidant compounds that are isolated from natural products.
