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Antiviral and antimicrobial activities of Paeonia lactiflora root constituents and structurally related compounds against human rhinovirus and gastrointestinal bacteria
적작약 뿌리 성분 및 구조적 유사 화합물의 human rhinovirus에 대한 항바이러스 활성 및 장내세균에 대한 항균활성

DC Field Value Language
dc.contributor.advisorAhn, Young-Joon-
dc.contributor.authorLuong, Thi My Ngan-
dc.date.accessioned2017-07-13T17:37:25Z-
dc.date.available2017-07-13T17:37:25Z-
dc.date.issued2013-02-
dc.identifier.other000000008966-
dc.identifier.urihttps://hdl.handle.net/10371/120975-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 협동과정 농업생물공학전공, 2013. 2. 안영준.-
dc.description.abstractAn assessment was made of the antiviral and antibacterial activities of paeony, Paeonia lactiflora Pallas (Paeoniaceae), root constituents and structurally related compounds against two serotypes of human rhinovirus, HRV-2 and HRV-4, in HeLa ATCC CCL–2 (a human cervix epithelial cell line) and MRC5 CCl-171 (a normal human lung fibroblast cell line) and antibiotic-susceptible and -resistant strains of Helicobacter pylori and human intestinal bacteria (nine harmful bacteria and eight lactic acid-producing bacteria). Modes of antiviral and antibacterial action also were examined using molecular and biochemical techniques.
The antiviral activities of 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose (PGG), paeonol (PA), and gallic acid (GA) identified in paeony root against HRV-2 and HRV-4 were evaluated using a tetrazolium assay. Results were compared with those of ribavirin, a broad spectrum antiviral agent. PA and ribavirin did not cause cytotoxicity to both cell lines (CC50, >500 μg/mL). PGG exhibited low toxicity to both cell lines (CC50, ~102 μg/mL). GA caused high toxicity to MRC5 (CC50, 37.0 μg/mL), whereas it caused very low toxicity to HeLa cells (CC50, 255.2 μg/mL). Based on fifty percent inhibitory concentration (IC50) values, PGG was the most active compound against the HRVs (10.9 and 13.53 μg/mL in HeLa cells
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dc.description.abstract16.83 and 16.31 μg/mL in MRC5 cells to HRV-2 and HRV-4, respectively), followed by GA (72.6 and 76.23 μg/mL in HeLa cells to HRV-2 and HRV-4, respectively). GA did not inhibit the viruses in the MRC5 cells. Ribavirin and PA exhibited high and moderate activity, respectively against the HRVs in both cell lines (IC50, 58‒79 μg/mL and 82‒101 μg/mL, respectively). PGG and PA inhibited RNA replication of HRV-2 and HRV-4 and also reduced expression of rhinovirus receptors and inflammatory cytokines induced by the two HRV serotypes. Quantitative structure–activity relationship (QSAR) of paeonol and 10 structurally related compounds in MRC5 cells indicates that structural characteristics, such as types of functional groups and carbon skeleton appear to play a role in determining the anti-rhinovirus activity. The introduction of methoxy group(s) into acetophenone may be required for rhinovirus inhibition.
The growth-inhibiting, bactercidial, and urease inhibitory activities of PA, benzoic acid (BA), methyl gallate (MG), and PGG identified in paeony root, structurally related compounds, root steam distillate constituents, and four antibiotics toward three reference strains and four clinical isolates of H. pylori were assessed using broth dilution bioassay and Western blot. BA and PA showed strong bactericidal effect at pH 4, while MG and PGG were effective at pH 7. These constituents exhibited strong growth-inhibiting and bactericidal activity toward the five strains resistant to amoxicillin (minimal inhibitory concentration (MIC), 12.5 mg/L), clarithromycin (64 mg/L), metronidazole (64 mg/L), or tetracycline (15 mg/L), indicating that these constituents and the antibiotics do not share a common mode of action or elicit cross-resistance. QSAR of paeonol and 10 structurally related compounds indicates that structural characteristics, such as types of functional groups and carbon skeleton, and hydrophobicity appear to play a role in determining the anti-H. pylori activity. H. pylori urease inhibitory activity of PGG was comparable to that of acetohydroxamic acid, while MG was less potent at inhibiting urease than thiourea. The UreB band disappeared at 250 mg/L PGG on Western blot, while the UreA bands were fainted visible at 1,000 mg/L PGG. These constituents showed no significant cytotoxicity to three human cell lines, HeLa, MRC5, and A549 (a human lung carcinoma cell line). High potent anti-H. pylori activity was also produced by steam distillate and its constituents. Thymol, α-terpinolene, (1R)-(‒)-myrtenol, (1S,2S,5S)-(‒)-myrtanol, (‒)-perilla alcohol, (‒)-borneol, (1R)-(‒)-myrtenal and paeonol showed high activity (MIC, 40–160 mg/L). The other constituents of the SD showed moderate or weak activity. The steam distillate exhibited strong growth-inhibiting and bactericidal activity against the five resistant strains indicating that these strains were lack of mechanisms of resistance to the steam distillate. In addition, the steam distillate inhibited H. pylori colonization in mouse stomach.
The growth inhibitory activities of paeony root steam distillate constituents and structurally related compounds against nine harmful intestinal bacteria and eight lactic acid-producing bacteria were compared with those of two antibiotics, amoxicillin and tetracycline. Thymol, α-terpinolene, (‒)-perilla alcohol, and (1R)-(‒)-myrtenol exhibited high to extremely high levels of growth inhibition of all the harmful bacteria, whereas thymol and α-terpinolene (except for Lactobacillus casei ATCC 393) inhibited the growth of all the beneficial bacteria (MIC, both 0.08–0.62 mg/mL). Tetracycline and amoxicillin exhibited extremely high level of growth inhibition of all the test bacteria (MIC, <0.00002–0.001 mg/mL). 1,8-Cineole, geraniol, (‒)-borneol, (1S,2S,5S)-(‒)-myrtanol, nerol, (S)-(‒)-β-citronellol, and (+/-)-lavandulol also exhibited inhibitory activity but with differing specificity and levels of activity. Structure–activity relationship indicates that structural characteristics, such as geometric isomerism, degrees of saturation, types of functional group and types of carbon skeleton, appear to play a role in determining the growth-inhibiting activity of monoterpenoids.
In conclusion, global efforts to reduce the level of antibiotics justify further studies on P. lactiflora root-derived materials as potential antiviral or antibacterial products, or lead molecules for the prevention or eradication of human diseases caused by rhinovirus, H. pylori, and harmful intestinal bacteria such as clostridia.
Keywords: Paeonia lactiflora root, natural growth inhibitor, natural bactericide, gatrointestinal bacteria, Helicobacter pylori, human rhinovius, antibiotic resistance, urease, terpenoids, structure– activity relationship
Student number: 2007 – 30696
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dc.description.tableofcontentsABSTRACT ………….………........………………………………… i
ABBREVIATIONS …………….........…….…………………………… x
LIST OF TABLES ………….…….........………………………………… xii
LIST OF FIGURES ……….……….........……………………………… xvi
INTRODUCTION ……….………........………………………………… 1
LITERATURE REVIEW ……………........……………………………… 4
Chapter 1. Antiviral activity of constituents identified in Paeonia lactiflora root against rhinovirus
Abstract 35
1.1. Introduction ………………………..…….….........………….………………… 37
1.2. Materials and methods ……..…………….........…………….……………… 38
1.2.1. Instrumental analyses 38
1.2.2. Materials 38
1.2.3. Plant 39
1.2.4. Cell lines and human rhinovirus serotypes 39
1.2.5. Cytotoxicity assay 41
1.2.6. HRV production 41
1.2.7. Virus titration 42
1.2.8. Cytopathic effect inhibition assay 43
1.2.9. Extraction and isolation 43
1.2.10. Effect of active constituents on the infectivity of human rhinovirus particles 48
1.2.11. Time course of compound addition 48
1.2.12. Real-time quantitative RT-PCR analysis 49
1.2.13. Measurement of ICAM-1 and LDLR expression 50
1.2.14. Western Blotting 52
1.2.15. Measurement of cytokine production 53
1.2.16. Data analysis 53
1.3. Results …………………..………………………………………………………… 55
1.3.1. Bioassay-guided fractionation and isolation of active principles 55
1.3.2. Cytotoxicity of test compounds 65
1.3.3. Antiviral activity of test compounds 66
1.3.4. Effects of PA and PGG on the infectivity of HRV particles 72
1.3.5. Time course of test compound addition 73
1.3.6. Effects of PA and PGG on the level of HRV replication 74
1.3.7 Effects of PA and PGG on ICAM-1 and LDLR expressions 75
1.3.8. Effects of PA and PGG on expression of cytokines IL-6 and IL-8 80
1.3.9. Effects of PA and PGG on expressions of other cytokines 84
1.3.10. Effects of PA and PGG on TLR3 mRNA expression 87
1.4. Discussion …..…………………………………………………………….............. 88
Chapter 2. Growth-inhibiting, bactericidal, and urease inhibitory effects of
Paeonia lactiflora root constituents on antibiotic-susceptible
and -resistant strains of Helicobacter pylori
Abstract 96
2.1. Introduction …………………………...………………………………...... 98
2.2. Materials and Methods ………………………………..…………........... 101
2.2.1. Chemicals 101
2.2.2. Bacterial strains and culture conditions 101
2.2.3. Steam distillation 102
2.2.4. Gas chromatography 102
2.2.5. Gas chromatography-mass spectrometry 102
2.2.6. Extraction and isolation 103
2.2.7. Microbiological assay 106
2.2.8. Measurement of bactericidal activity at various pH values 107
2.2.9. Microscopic observation 108
2.2.10. Scanning and transmission electron microscopic analysis 108
2.2.11. Cytotoxicity assay 109
2.2.12. Inhibition of urease in vitro 109
2.2.13. SDS-PAGE and Western blot analysis 110
2.2.14. Native PAGE and zymogram analysis 111
2.2.15. In vivo study 111
2.2.16. Data analysis 113
2.3. Results …………………………………...………………………................ 115
2.3.1. Antibiotic resistance 115
2.3.2. Paeonia lactiflora root steam distillate 117
2.3.2.1. Chemical composition of P. lactiflora root steam distillate 117
2.3.2.2. Growth-inhibiting activities of P. lactiflora root steam distillate constituents 119
2.3.2.3. Bactericidal activities of P. lactiflora root steam distillate constituents 119
2.3.2.4. Effect on morphology of H. pylori 123
2.3.2.5. Effects of P. lactiflora root steam distillate on H. pylori colonization in mouse stomach 124
2.3.3. Paeonia lactiflora root extract 125
2.3.3.1. Bioassay-guided fractionation and identification 125
2.3.3.2. Growth inhibitory activity of test compounds 133
2.3.3.3. Bactericidal activity of test compounds 134
2.3.3.4. Effect of test compounds on the viability of H. pylori at varying pH values 136
2.3.3.5. Structure–activity relationship 142
2.3.3.6. Cytotoxicity 145
2.3.3.7. Effect on morphology of H. pylori 145
2.3.3.8. Urease inhibitory activity 147
2.3.3.9. Effect on urease production 148
2.4. Discussion ………………………………...…………………………… 151

Chapter 3. Growth-inhibiting effects of Paeonia lactiflora root steam distillate constituents and structurally related compounds
on human intestinal bacteria
Abstract 156
3.1. Introduction ……………………………………………………..........… 157
3.2. Materials and Methods …………...……………………………….......... 159
3.2.1. Steam distillation 159
3.2.2. Chemicals 159
3.2.3. Bacterial strains and culture conditions 159
3.2.4 Growth-inhibiting assay 161
3.3. Results …………………………………………………………………… 162
3.4. Discussion ………………...……………………………………………… 170
Conclusion .................................................................................................. 175
References ................................................................................................... 180
Abstract in Korean ..................................................................................... 219
Acknowledgement .................................................................................. 224
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dc.formatapplication/pdf-
dc.format.extent3323112 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectPaeonia lactiflora root-
dc.subjectnatural growth inhibitor-
dc.subjectnatural bactericide-
dc.subjectgatrointestinal bacteria-
dc.subjectHelicobacter pylori-
dc.subjecthuman rhinovius-
dc.subjectantibiotic resistance-
dc.subjecturease-
dc.subjectterpenoids-
dc.subjectstructure– activity relationship-
dc.subject.ddc660-
dc.titleAntiviral and antimicrobial activities of Paeonia lactiflora root constituents and structurally related compounds against human rhinovirus and gastrointestinal bacteria-
dc.title.alternative적작약 뿌리 성분 및 구조적 유사 화합물의 human rhinovirus에 대한 항바이러스 활성 및 장내세균에 대한 항균활성-
dc.typeThesis-
dc.contributor.AlternativeAuthorLuong Thi My Ngan-
dc.description.degreeDoctor-
dc.citation.pages255-
dc.contributor.affiliation농업생명과학대학 협동과정 농업생물공학전공-
dc.date.awarded2013-02-
Appears in Collections:
College of Agriculture and Life Sciences (농업생명과학대학)Program in Agricultural Biotechnology (협동과정-농업생물공학전공)Theses (Ph.D. / Sc.D._협동과정-농업생물공학전공)
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