An Exploratory Study on the Antioxidant, NO Inhibitory and Antiproliferative Activities of Crude Extract and Its Solvent Fractions of the Halophyte, Angelica japonica, Growing in Korean Coastal Area

The Halophyte, Angelica japonica is an edible, perennial plant and traditionally has been used for several medicinal purposes. Crude extract and its solvent fractions of A. japonica were investigated for antioxidizing ability, cancer cell anti-proliferation, and inhibition of NO production. Antioxidant activity screening was performed by measuring scavenging effect of DPPH radical, peroxynitrite (ONOO) and intracellular ROS (reactive oxygen species), genomic DNA damage, and ferric-reducing power. Also these antioxidant results were confirmed by polyphenol and flavonoid contents analysis. NO inhibitory effect was evaluated by measurement of nitric oxide production in macrophage Raw 264.7 cells. Antiproliferative activity screening was run by measuring cell viability of human fibrosacoma (HT-1080), human gastric adenocarcinoma (AGS), human colon tumor (HT-29), human breast cancer (MCF-7) cell lines using MTT assay. Also Western blot MMP-2 and MMP-9, and cell migration assays for HT 1080 cells have done. Plant materials of A. japonica were collected by hand at Jeju Island and air-dried in shade. The dried plant of A. japonica was extracted twice with methyl chloride and twice with methanol in turn and then crude extract obtained after removal of extract solvent was fractionated into n-hexane, 85% aqeous methanol (85% aq.MeOH), n-butanol, and water. Overall, significant antioxidant effects were successfully observed in crude extract and its solvent fractions (n-BuOH and 85% aq.MeOH fractions). For DPPH radical scavenging activity, crude extract and n-BuOH fraction showed a slight scavenging effect, compared to other fractions but didn’t show a significant scavenging effect, compared to BHA, BHT and vitamin C. For peroxynitrite, crude extract, and n-BuOH and 85% aq.MeOH fractions showed high scavenging effects in a concentration-dependent manner. In an experiment to measure effect of scavenging ROS generated in Raw 264-7 cells, crude extract, and 85% aq.MeOH and n-BuOH fractions exhibited high scavenging ability. In case of NO production, crude extract showed an inhibitory effect against nitrite oxide (NO) produced in Raw 264.7 cells stimulated with lipopolysaccharide (LPS) in a dose-dependent way. But all solvent fractions didn’t show the significant inhibitory effect against NO generation. In proliferation inhibition test for cancer cells, all solvent fractions showed concentration-dependently good antiproliferative effects on all human cancer cell lines used (HT -1080, AGS, HT-29 & MCF-7), especially the effect was high in MCF-7 cell line, and the 85% aq. MeOH fraction revealed relatively the high effect. n-BuOH fraction showed good inhibitory effect against HT-1080 cell line. In addition, inhibitory effect of crude extract and its solvent fractions on MMP-9 and MMP-2 was evaluated in human fibrosarcoma cell line (HT-1080). The n-hexane and 85% aq.MeOH fractions remarkably reduced expression levels on MMP-9 and MMP-2. Examining antioxidant and antiproliferative activities mention above, all the activities were observed in a concentration-dependent manner, and n-BuOH and 85% aq.MeOH fractions showed the best activity among solvent fractions. However, the crude extract and the solvent fractions did not show significant activity for NO production inhibition. Since polyphenol content analysis showed that the n-BuOH and 85% aq.MeOH fractions contained high polyphenol content, the antioxidant and antiproliferative effects of these solvent fractions may be partially due to polyphenols. Therefore, these results suggest that A. japonica may be a good source for development of antioxidants and anticancer drugs.1. Introduction 1 1.1. Halophytes 1 1.2. Antioxidant Activity 3 1.3. NO Inhibitory Activity 3 1.4. Antiproliferative Activity 4 1.5. Objectives 5 2. Materials and Methods 6 2.1. Plant Material 6 2.2. General Experimental Procedures 7 2.2.1. Chemicals and Reagents 7 2.3. Extraction and Fractionation of Angelica japonica 8 2.4. Antioxidant Activity 10 2.4.1. DPPH Radical Scavenging Activity 10 2.4.2. Peroxynitrite Scavenging Activity 13 2.4.3. Ferric Reducing Antioxidant Power (FRAP) Assay 16 2.4.4. Determination of Intracellular Formation of Reactive Oxygen Species(ROS) Using DCFH-DA Labelling 17 2.4.5. Genomic DNA Extraction and Determination of Radical Mediated DNA Damage 19 2.4.6. Determination of Total Polyphenol Content (TPC) 20 2.4.7. Determination of Total Flavonoid Contents ` 21 2.5. NO inhibitory Activity 22 2.5.1. Cell Culture and Measurement of Cell Viability by MTT Assay against Raw 264.4 Cells 22 2.5.2. Determination of Nitric Oxide (NO) Production Using Macrophage Raw 264.4 Cells 23 2.6. Anti-proliferative Activity against Human Cancer Cells 25 2.6.1. Cell Cultures and Inhibition of Cancer Cell Proliferation 25 2.6.2. Effect of Crude Extract and its Solvent Fractions on Protein Levels of MMP-9 and MMP-2 by Western Blot Assay 27 2.6.3. Cell Migration Assay Observation Using the Wound Healing Assay 28 2.7. Statistical Analysis 29 3. Results and Discussion 30 3.1. Evaluation of Antioxidant Effects in Crude Extract and Its Solvent Fractions of Angelica japonica 30 3.1.1. Scavenging Activity on DPPH Radicals 30 3.1.2. Peroxynitrite Scavenging Activity 32 3.1.3. Ferric Reducing Antioxidant Power (FRAP) Assay 34 3.1.4. Determination of Intracellular Formation of Reactive Oxygen Species (ROS) Using DCFH-DA Labelling 35 3.1.5. Determination of Total Polyphenol Content (TPC) & Total Flavonoid Content (TFC) 40 3.1.6. Genomic DNA Extraction and Measurement Genomic DNA Oxidation 41 3.2. NO Inhibitory Effects of Crude Extract and Its Solvent Fractions 42 3.2.1. Measurement of Cell Viability by Crude Extract and Its Solvent Fractions in Raw 264.7 Cells 42 3.2.2 Determination of Inhibitory Effect on Nitric Oxide (NO) Production 43 3.3. Antiproliferative Effects against the Growth of Human Cancer Cells 45 3.3.1. Cytotoxic Effect against Human Cancer Cells 45 3.3.2. Inhibitory Effect against Expression of Protein Levels of MMP- 9 and MMP-2 by Western Blot Assay 48 3.3.3. Effect of Crude Extract and Its Solvent Fractions on Cell Migration Ability by the Wound Healing Assay 49 4. Conclusion 51 5. References 54Maste