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제안 방법
- However, studies reporting upon the activities of dangyuja leaves are limited. In this study we propose to evaluate the potential antioxidant and cytoprotective effects of ethyl acetate fraction (EF) of dangyuja leaves against tert-butyl hydroperoxide (t-BHP) induced oxidative damages in HepG2 cells. This hepatoma cell line is considered to be a good tool to study the cytoprotective effects of compounds or natural antioxidants to liver cells (18,19).
- Therefore, we investigated the antioxidant properties of the EF in a series of assays such as intracellular ROS generation, lipid peroxidation, DNA damage, and cytotoxicity using this model of in vitro hepatotoxicity. Furthermore, because it is important to determine the constituents present in the extract which may be contributing to the activity, a compositional analysis of the EF was carried out using gas chromatography-mass spectroscopy (GC-MS).
- Gas chromatography GC analysis of EF was carried out with Aligent 6850 system equipped with a flame ionization detector (FID) and HP-5MS capillary column (30 m×0.25 mm i.d., 0.25 µm film thickness).
- Determination of total phenolics and flavonoids Total phenolics were determined according to the method of Cheung et al. (21) with slight modification. Two mL of sample were mixed with 2 mL of Folin-Ciocalteu phenol reagent (1:1 with water).
- GC-MS GC-MS analysis of EF was carried out with Agilent 6890N gas chromatography coupled to Agilent 5975N mass spectrometer. GC conditions and column were the same as those used in the GC analysis above.
대상 데이터
- Materials Dangyuja leaves were collected from the National Institute of Subtropical Agriculture, Jeju, Korea. Botanical samples were taxonomically identified by Kim (20) and a voucher specimen (No.
- 070531) is deposited at the laboratory of Professor Somi Kim Cho in the Faculty of Biotechnology, College of Applied Life Sciences, Cheju National University. All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Invitrogen Gibco (Grand Island, NY, USA).
- Cell culture Human hepatoma HepG2 (KCLB No. 58065) cells were purchased from the Korea Cell Line Bank (Seoul, Korea). The cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10%(v/v) heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin.
이론/모형
- Determination of ROS generation Cellular ROS were quantified using the dichlorofluorescein (DCFH) assay (24), in which the esterified form of DCFH-diacetate diffuses through the cell membrane and is enzymatically deacetylated by intracellular esterases. The resulting compound, DCFH, is reactive with ROS to give an oxidized fluorescent compound, DCF (25).
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