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Title Page

국문초록

Contents

ABBREVIATIONS 12

I. INTRODUCTION 13

1. Inflammation 13

2. Antioxidation 15

3. Agrimonia pilosa Ledeb 17

II. MATERIALS AND METHODS 18

1. Materials 18

1) Plant 18

2) Cell culture 18

3) Experimental animal 18

2. Methods 19

1) Extraction procedure 19

2) Quantification of flavonoid 20

3) Measurement of cell viability by MTT assay 20

4) Measurement of free radical scavenging activity 20

5) TLC (Thin Layer Chromatography) analysis 21

6) HPLC (High Performance Liquid Chromatography) analysis 21

7) Antioxidative activity 23

8) Effect of Agrimonia pilosa ethyl acetate fraction in vitro 24

9) Antioxidative effect of Agrimonia pilosa ethyl actate fraction on LPS-induced liver injury in vivo 28

3. Statistical analysis 32

III. RESULTS AND DISCUSSION 33

1. Flavonoid content, antioxidant capacity and cytotoxicity of five solvent fractions from Agrimonia polisa ethanol extract. 33

2. Component analysis of Agrimonia pilosa ethyl acetate fraction using TLC and HPLC 37

1) Component analysis of Agrimonia pilosa ethyl acetate fraction using TLC 37

2) Quercitrin quantification of Agrimonia pilosa ethyl acetate fraction using HPLC 39

3. Effect of Agrimonia pilosa ethyl acetate fraction on antioxidative activity 41

1) Superoxide radical scavenging activity of Agrimonia pilosa ethyl acetate fraction 41

2) Hydroxyl radical scavenging activity of Agrimonia pilosa ethyl acetate fraction 43

4. Effect of Agrimonia pilosa ethyl actate fraction in vitro 45

1) LPS-induced cytotoxicity and determination of LD50 value in MTT assay(이미지참조) 45

2) The protective effects of Agrimonia pilosa ethyl acetate fraction on cytotoxicity of the value of LD50 (10.53㎍/㎖ ofLPS) in MTT assay(이미지참조) 47

3) Effect of Agrimonia pilosa ethyl acetate fraction on LPS-produced NO in RAW 264.7 macrophages 49

4) Effect of Agrimonia pilosa ethyl acetate fraction on LPS-produced PGE₂ in RAW 264.7 macrophages 51

5) The protective effect of Agrimonia pilosa ethyl acetate fraction on LPS (LD50＝10.53㎍/㎖) genotoxicity in cometassay(이미지참조) 53

5. Effect of Agrimonia pilosa ethyl acetate fraction in vivo 57

1) Effects of Agrimonia pilosa ethyl acetate fraction on bioactivity enzymes in sera of LPS-induced rats 57

2) Effect of Agrimonia pilosa ethyl acetate fraction on lipid peroxidation of LPS-induced rats 59

3) Effects of Agrimonia pilosa ethyl acetate fraction on oxidation of LPS-induced rats 61

4) Effect of AE on DNA damage in lymphocyte of LPS-induced rats 67

IV. CONCLUSION 70

V. REFERENCES 72

ABSTRACT 79

List of Tables

Table 1. Analytical conditions of HPLC for quercitrin of Agrimonia pilosa ethyl acetate fraction 22

Table 2. Experimental design of lipopolysaccharide-induced rats 29

Table 3. Flavonoid content of five fractions from Agrimonia pilosa ethanol extract 34

Table 4. DPPH radical scavenging effect of five fractions from Agrimonia pilosa Ledeb. ethanol extract 35

Table 5. Scavenging effect of Agrimonia pilosa ethyl acetate fraction on superoxide radical 42

Table 6. Scavenging effect of Agrimonia pilosa ethyl actate fraction on Hydroxyl radical 44

Table 7. The protective effect of Agrimonia pilosa ethyl acetate fraction against LPS-induced genotoxicity in tail parameters analysis 56

Table 8. Levels of AST, ALT and LDH in sera 58

Table 9. Levels of MDA in liver and kidney homogenates 60

Table 10. Levels of SOD in liver and kidney homogenates 64

Table 11. Levels of CAT in liver and kidney homogenates 65

Table 12. Levels of GPx in liver and kidney homogenates 66

Table 13. Effect of Agrimonia pilosa ethyl acetate fraction on DNA damage of lymphocytes of LPS-induced rats in tail parameter analysis 69

List of Figures

Fig. 1. Production of free radicals caused by various factor 16

Fig. 2. Fraction scheme of Agrimonia polisa Ledeb. ethanol extract 19

Fig. 3. Cytotoxicity of five fractions isolated from Agrimonia pilosa ethanol extract determined by MTT assay 36

Fig. 4. TLC analysis of quercitrin and kaempferol in Agrimonia pilosa ethyl acetate fraction 38

Fig. 5. HPLC analysis of Agrimonia pilosa ethyl acetate fraction 40

Fig. 6. The cytotoxicity of LPS and determination of LD50 value in RAW 264.7 macrophages(이미지참조) 46

Fig. 7. The protective effects of Agrimonia pilosa ethyl acetate fraction against LPS-induced cytotoxicity in RAW 264.7 macrophages by MTT assay 48

Fig. 8. Effect of Agrimonia pilosa ethyl acetate fraction on LPS-produced NO in RAW 264.7 macrophages 50

Fig. 9. Effect of Agrimonia pilosa ethyl acetate fraction against LPS-produced PGE₂ by PGE₂ assay 52

Fig. 10. The protective effect of Agrimonia pilosa ethyl acetate fraction against LPS-induced genotoxicity in the comet image analysis 54

Fig. 11. Effects of Agrimonia pilosa ethyl acetate fraction on DNA damage of blood lymphocytes in comet image analysis 68

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