Title Page
Contents
CHAPTER 1. Introduction 13
1. Biofilm 17
2. Dental biofilm of Porphyromonas gingivalis 21
3. Virulence factor of Porphyromonas gingivalis 22
4. Periodontal diseases 26
5. Diospyros malabarica 28
CHAPTER 2. Isolation and identification of putative antimicrobial compounds from Diospyros malabarica stems 29
ABSTRACT 30
INTRODUCTION 31
MATERIALS AND METHODS 35
1. Bacterial strains and culture conditions 35
2. Fractionation of crude extracts 37
3. Antimicrobial activity 39
4. Separation and purification of putative antimicrobial compounds 41
5. Identification of putative antimicrobial compounds 43
RESULTS 45
1. Antimicrobial activity 45
2. Separation and purification of putative antimicrobial compounds 51
3. Identification of putative antimicrobial compounds 56
DISCUSSION 77
CHAPTER 3. Anti-biofilm activity of compound MF-1 against Porphyromonas gingivalis 80
ABSTRACT 81
INTRODUCTION 83
MATERIALS AND METHODS 87
1. Culture conditions 87
2. Quantitative determination of biofilm 87
3. Scanning electron microscopy (SEM) 88
4. Transmission electron microscopy (TEM) 89
5. Expression of biofilm-associated gene 90
6. Statistical analysis. 90
RESULTS 92
1. Effects of crude extracts on colony morphology of Porphyromonas gingivalis of on a congo red agar plate 92
2. Quantitative determination of biofilm 94
3. Scanning electron microscopy (SEM) 96
4. Observation of fimbriae by TEM 98
5. Expression of biofilm-associated gene 100
DISCUSSION 104
CHAPTER 4. Anti-inflammatory activity of compound MF-1 107
ABSTRACT 108
INTRODUCTION 109
MATERIALS AND METHODS 113
1. Cell culture 113
2. Cell viability of crude extracts and compound MF-1 114
3. Nitric oxide (NO) assay 115
4. Expression of gingipain-associated gene 116
5. Statistical analysis 118
RESULTS 119
1. Cell viability of crude extracts and compound MF-1 from Diospyros malabarica stems 119
2. Nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells 121
3. Expression of gingipain-associated gene 123
DISCUSSION 126
REFERENCES 128
ABSTRACT (국문) 147
Table 1. Bacterial strains and culture conditions used in this study 36
Table 2. MIC of crude extracts from Diospyros malabarica stems against... 46
Table 3. MBC of crude extracts from Diospyros malabarica stems against... 46
Table 4. Experimental conditions for the HPLC analysis 54
Table 5. Crystal datas and structure refinement for compound MF-1 64
Table 6. Atomic coordinates (x10⁴) and equivalent isotropic displacement... 66
Table 7. Bond lengths [Å] and angles [°] for compound MF-1 68
Table 8. Anisotropic displacement parameters (Ųx10³) for compound MF-1 71
Table 9. Hydrogen coordinates (x10⁴) and isotropic displacement parameters... 73
Table 10. Characteristics of compound MF-1 and MF-2 75
Table 11. Oligonucleotide sequence of fimbriae-associated genes used in this study 91
Table 12. Oligonucleotide sequence of gingipain-associated genes used in this study 117
Fig. 1. Baterial cell-to-cell interactions in the dental plaque biofilm. 16
Fig. 2. Scheme of biofilm formation. 20
Fig. 3. Fimbriae of Porphyromonas gingivalis. 24
Fig. 4. Structures and homologies of gingipains associated gene rgpA, rgpB,... 25
Fig. 5. Biologically mechanisms associating periodontitis with systemic... 27
Fig. 6. Fractionation scheme of crude extracts from Diospyros malabarica stems. 38
Fig. 7. Antimicrobial activities of partitioned fraction (1 mg/disc) with solvents of... 48
Fig. 8. Antimicrobial activity of crude extracts and putative antimicrobial... 50
Fig. 9. TLC chromatogram of crude extracts (A) and putative antimicrobial... 52
Fig. 10. HPLC chromatogram of n-hexane fraction of crude extracts from... 55
Fig. 11. The molecular weight of compound MF-1 using EI-MS/MS. 57
Fig. 12. 500 MHz ¹H-NMR spectrum of compound MF-1 in CDCl₃. 59
Fig. 13. 125 MHz ¹³C-NMR spectrum of compound MF-1 in CDCl₃. 60
Fig. 14. 500 MHz ¹H-NMR spectrum of compound MF-2 in CDCl₃. 61
Fig. 15. 125 MHz ¹³C-NMR spectrum of compound MF-2 in CDCl₃. 62
Fig. 16. Structures of compound MF-1 (A) and MF-2 (B). 74
Fig. 17. GC-MS fragments of compound MF-2. 76
Fig. 18. Schematics of Porphyromonas gingivalis fimbriae. 86
Fig. 19. Colony morphology with (A) and without (B) crude extracts from... 93
Fig. 20. Bacterial growth and biofilm formation of Porphyromonas gingivalis... 95
Fig. 21. Scanning electron microscopy (SEM) micrographs of Porphyromonas... 97
Fig. 22. Transmission electron microscopy (TEM) micrographs of... 99
Fig. 23. Effects of crude extracts and compound MF-1 from Diospyros... 101
Fig. 24. Effects of crude extracts and compound MF-1 from Diospyros... 102
Fig. 25. Effects of crude extracts and compound MF-1 from Diospyros... 103
Fig. 26. Cell viability of crude extracts and compound MF-1 from Diospyros... 120
Fig. 27. Effects of crude extracts and compound MF-1 from Diospyros malabarica... 122
Fig. 28. Effects of crude extracts and compound MF- from Diospyros... 124
Fig. 29. Effects of crude extracts and compound MF-1 from Diospyros... 125