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Title Page

Abstract

Contents

I. INTRODUCTION 11

II. MATERIALS AND METHODS 14

A. Fractionation, isolation, and identification of bioactive component(s) 14

B. Reagents and cell culture materials 17

C. Water-soluble tetrazolium salt (WST) assay in osteoblast cells 18

D. Alkaline phosphatase (ALP) activity assay and staining in osteoblast cells 18

E. Mineralized nodule formation in osteoblast cells 19

F. Quantitative reverse-transcription PCR (qRT-PCR) 20

G. Osteoclastogenesis of primary cultured monocytes 21

H. Tartrate-resistant acid phosphatase (TRAP) activity assay and staining in monocytes 22

I. Co-culture of MC3T3-E1 cells and primary monocytes 22

J. Ovariectomized model 23

K. Measurement of bone mineral density (BMD) 24

L. Microcomputed tomography (Micro CT) and SPECT scan 24

M. Blood sampling and serum OPG and RANKL measurement 25

N. Statistical analysis 26

III. RESULTS 27

A. Cellular differentiation of osteoblastic ME3T3-E1 cells 27

B. Bioactive component of the Cornus officinalis extract 29

C. Effect of Loganin on osteoblast differentiation and mineralized nodule formation 31

D. Effect of Loganin on the expression of bone remodeling markers 33

E. Effect of Loganin on osteoclast differentiation 35

F. Effect of Loganin on osteoblast and osteoclast differentiation 37

G. Effect of Loganin on OVX-induced bone loss in an osteoporotic mouse model 39

H. Effect of Loganin on serum OPG and RANKL levels 45

IV. DISCUSSION 47

V. Conclusion 51

REFERENCES 52

국문요약 59

List of Tables

Table 1. Alkaline phosphatase (ALP) activity of MC3T3-E1 cells treated with... 28

List of Figures

Figure 1. Bone remodeling 13

Figure 2. Isolation of Loganin through the fractionation of a 70% ethanol... 15

Figure 3. Nuclear magnetic resonance spectra of the bioactive (D2) fraction. 16

Figure 4. Chemical structure of Loganin. 30

Figure 5. Effect of Loganin on the alkaline phosphatase (ALP) activity, ALP... 32

Figure 6. Effect of Loganin on the expression of bone remodeling markers. 34

Figure 7. Effects of Loganin on osteoclast differentiation in primary-cultured... 36

Figure 8. Effect of Loganin on the alkaline phosphatase (ALP) and tartrate-... 38

Figure 9. Effects of Loganin on the bone mineral density (BMD) of... 41

Figure 10. Anti-osteoporosis effect of Loganin on the bones of ovariectomized... 42

Figure 11. Anti-osteoporosis effect of Loganin on the femurs of ovariectomized... 43

Figure 12. Anti-osteoporosis effect of Loganin in ovariectomized (OVX) mice. 44

Figure 13. Anti-osteoporosis effect of Loganin on serum levels of OPG and... 46