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Chemical profiling of Farfarae Flos sesquiterpenoids and the target protein identification of an oplopane sesquiterpenoid in breast cancer cells : 관동화 유래 세스퀴테르펜 화합물의 성분프로파일 및 유방암 세포주에서의 oplopane 세스퀴테르펜의 표적 단백질에 관한 연구
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 김영식 | - |
| dc.contributor.author | 송광호 | - |
| dc.date.accessioned | 2019-10-21T03:11:51Z | - |
| dc.date.available | 2021-09-23T07:04:08Z | - |
| dc.date.issued | 2019-08 | - |
| dc.identifier.other | 000000156811 | - |
| dc.identifier.uri | https://hdl.handle.net/10371/162216 | - |
| dc.identifier.uri | http://dcollection.snu.ac.kr/common/orgView/000000156811 | ko_KR |
| dc.description | 학위논문(박사)--서울대학교 대학원 :약학대학 약학과,2019. 8. 김영식. | - |
| dc.description.abstract | 관동(Tussilago farfara L.)은 국화과의 다년생 약초로서 관동의 말린 꽃봉오리(관동화, Farfarae Flos)는 전통 의학에서 기침, 기관지염 및 천식과 같은 호흡기 질환을 치료하기 위해 사용되었다. 주요 생리활성성분으로는 플라보노이드, 테르페노이드, 퀸산유도체 등이 보고되었으며, 특히 세스퀴테르펜 화합물군이 항염증, 세포증식억제, 뇌신경보호 등에서 높은 효능을 나타내었다. 따라서 본 연구에서는 관동화 유래 세스퀴테르펜 화합물에 대한 1) 대량 분리법, 2) LC-MS 기반의 성분프로파일링, 3) 유방암 세포주에서의 표적단백질 규명을 수행하였다.
항류크로마토그래피를 이용하여 관동화 유래 세스퀴테르펜 화합물의 대량분획법(직접연속주입법, Direct and Continuous Injection mode)을 고안하였다. 추출액 자체를 이동상으로 사용함으로써 유기용매의 사용량을 크게 줄였으며, 관동화 1 kg의 추출물 315.9 g으로부터 6.8 g의 세스퀴테르펜 강화분획을 한 번에 획득하였다. 기존의 항류크로마토그래피 분리방법은 1~5 g의 추출물을 주입하기 때문에 직접연속주입법을 통해 분리 시간과 비용을 절감할 수 있었다. 또한, 주요 세스퀴테르펜 단일화합물의 정량분석을 수행하였고 용매분획법이나 컬럼크로마토그래피를 이용해 얻어진 분획물에 비해 높은 함량을 확인하였다. UPLC-MS/MS 기법을 이용하여 관동화의 oplopane 및 bisabolane 계열의 세스퀴테르펜에 대한 성분프로파일을 제시하였다. 해당 화합물군은 질량분석기의 ESI 이온화과정에서 쉽게 In-source fragmentation (IS-CID) 되는 화합물로서 모분자 질량값을 얻기 위해 QqQ-MS의 Precursor ion scan을 적용하였다. 구조적 특성을 기반으로 네 가지의 특이적 이온(diagnostic ions, [M+H]+ 215, 217, 229, 231)을 선정하여 총 74종의 화합물에 대한 모분자 질량값을 확인하였고, Q-TOF MS의 Product ion scan을 이용하여 각 모분자 이온의 특징적인 쪼개짐 양상(fragmentation pattern)을 고분해능 수준에서 확인하였다. 또한 11종의 화합물을 분리 및 구조동정하여 고안된 동시분석법을 검증하였고, MRMHR 분석법을 이용하여 관동화 추출물에 함유된 주요 세스퀴테르펜 화합물의 함량을 확인하였다. 유방암 세포주 MDA-MB-231과 MCF-7에 대한 ECN (7β-(3´-ethyl cis-crotonoyloxy)-1α-(2´-methyl butyryloxy)-3,14-dehydro-Z-notonipetranone)의 높은 세포증식억제능을 확인하였고, Chemical Proteomics 기법을 이용하여 표적단백질을 제시하였다. In vitro 스크리닝 결과, 관동화 유래 세스퀴테르펜 화합물군은 항염증효능 뿐만 아니라 세포증식억제능을 보였으며 그 중 ECN이 가장 높은 효능을 나타냈다. ECN 기반의 clickable probe를 합성하여 세포 내에서의 click 반응으로 표적 단백질을 분획하였고, 가수분해된 펩타이드 혼합물의 TMT isobaric label 및 Orbitrap MS/MS 분석을 통하여 음성대조군 대비 3배 이상의 선택성을 갖는 17종의 표적단백질을 규명하였다. 또한, 높은 선택성을 갖는 두 종의 표적단백질 14-3-3 protein zeta, peroxiredoxin-1에 대한 ECN의 작용 위치(binding site)와 결합친화도(ITC)를 확인하였다. 본 연구 결과들을 종합하여 볼 때, 관동화 유래 세스퀴테르펜 화합물군에 대한 LC-MS/MS 성분프로파일은 관동화를 포함하는 생약제제의 품질관리에 적용될 수 있으며, oplopane 골격의 세스퀴테르펜에 대한 표적단백질을 규명함으로써 세포증식억제능에 한하여 유효성분과 약효의 상관관계를 확인하는데 기초가 되는 연구로 사료된다. | - |
| dc.description.abstract | Farfarae Flos is the dried buds of Tussilago farfara L., a perennial plant of the family Asteraceae, and has been used to treat coughs, bronchitis, and asthmatic conditions in traditional herbal medicine. Among its bioactive compounds, sesquiterpenoids exhibit various biological activities such as anti-inflammative, anti-proliferative, and neuroprotective effects. In the present study, preparative separation, chemical profiling, and activity-based proteome profiling of sesquiterpenoids from Farfarae Flos were performed.
Firstly, a novel fractionation and purification method of counter-current chromatography (CCC), called direct and continuous injection (DCI) mode, was developed to fractionate and preparatively separate sesquiterpenoids from Farfarae Flos. Since the extraction solution was used as a mobile phase in this method, solvent consumption was greatly reduced. 6.8 g of sesquiterpenoid-enriched (STE) fraction was obtained from the crude extract (315.9 g) of Farfarae Flos (1 kg) in a single CCC run with a separation time of 8.5 hrs. The sample injection capacity of CCC-DCI was greater than 300 grams which could not be handled in conventional CCC methods. Moreover, quantification study indicated that the fractionation efficiency of CCC-DCI was higher than those of conventional fractionations: solvent partitioning and open column chromatography. The developed method demonstrates that CCC is a useful technique for enriching target components from natural products. Secondly, a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)-based dereplicative method was developed to identify and quantify oplopane- and bisabolane-type sesquiterpenoids of Farfarae Flos. The MS-based nontargeted metabolomic approach for these chemical analogues, sesquiterpene esters, is challenging because of their in-source fragmentation and structural diversity. In order to profile these sesquiterpenoids, four diagnostic ions (m/z 215.143, 217.158, 229.123, and 231.138) were suggested in the positive ion mode and the developed method utilized two sequential MS/MS scan modes to characterize common skeletons and investigate the fragmentation patterns of their parent molecules. Under the optimized UHPLC-MS/MS method, 74 sesquiterpenoids were identified from the Farfarae Flos and 11 compounds were isolated for the method validation. Furthermore, the diagnostic ions and the MS/MS fragment behaviors were applied to accurate quantification of the 8 isolated sesquiterpenoids. Consequently, the developed LC-MS/MS-based dereplicative method highlighted the chemical composition of the Farfarae Flos and could be applied to quality control of the herbal medicine. Finally, target proteins of ECN (7β-(3´-ethyl cis-crotonoyloxy)-1α-(2´-methyl butyryloxy)-3,14-dehydro-Z-notonipetranone) in human breast cancer cells were identified by chemical proteomics methodology. ECN showed potent anti-proliferation activity in MDA-MB-231 and MCF-7 human breast cancer cells based on its α,β-unsaturated carbonyl moiety. Therefore, the potential cellular target proteins of ECN were identified using ECN-based clickable probe and quantitative MS/MS analysis. Among more than 200 identified proteins, 17 proteins showed more than 3 enrichment ratio in both cell lines. Furthermore, recombinant 14-3-3 protein zeta and peroxiredoxin-1 were further verified by isothermic calorimetry and their alkylation sites. Taking the interaction between α,β-unsaturated carbonyl moiety of ECN and cysteine residues of proteins into account, peptides containing Cys25, Cys94 of 14-3-3 protein zeta and Cys83 of peroxiredoxin-1 were significantly reduced by ECN. Although these results could not confirm the role of ECN in the breast cancer cells, this suggestion of multiple target proteins contributed to understand the ECN-mediated anti-proliferative and anti-inflammatory effects, leading to further studies. | - |
| dc.description.tableofcontents | CONTENTS
ABSTRACT ...................................................................................................... I CONTENTS ................................................................................................... IV LIST OF FIGURES ................................................................................... IX LIST OF TABLES ..................................................................................... XII I. INTRODUCTION .................................................................................... 1 1. Farfarae Flos .................................................................................................. 2 1.1. Constituents and bioactivities ................................................................... 2 1.2. Oplopane and bisabolane sesquiterpenoids ............................................. 4 2. Counter-current chromatography (CCC) .............................................. 6 2.1. Background ................................................................................................. 6 2.2. Solvent selection and application .............................................................. 9 3. Chemical profiling ...................................................................................... 12 3.1. Liquid chromatography and mass spectrometry (LC-MS) .................. 12 3.2. Scan modes of tandem mass spectrometry (MS/MS) ............................ 14 3.3. LC-MS/MS-based dereplication methodology ...................................... 17 4. Activity-based proteome profiling .......................................................... 20 4.1. Background ............................................................................................... 20 4.2. Click chemistry ......................................................................................... 22 4.3. Quantitative proteome profiling based on mass spectrometry ............ 24 II. STATE OF THE PROBLEM .......................................................... 26 III. MATERIALS AND METHODS ................................................. 29 1. Materials ....................................................................................................... 30 1.1. Farfarae Flos ............................................................................................. 30 1.2. Chemicals and reagents ........................................................................... 30 1.3. Apparatus .................................................................................................. 31 1.4. Cell lines .................................................................................................... 32 2. Methods ......................................................................................................... 33 2.1. An efficient fractionation method for the preparative separation of sesquiterpenoids from Farfarae Flos by CCC ........................................ 33 2.1.1. Measurement of the partition coefficient (KD) ................................. 33 2.1.2. Preparation of the extract solution and solvent system ................... 33 2.1.3. CCC-DCI fractionation ...................................................................... 34 2.1.4. Solvent partitioning and open column chromatography ................ 35 2.1.5. Isolation of three major sesquiterpenoids ........................................ 36 2.1.6. Preparation of sample solution .......................................................... 36 2.1.7. HPLC analysis and calibration curve ............................................... 37 2.2. Chemical profiling of sesquiterpenoids from Farfarae Flos based on LC-MS/MS analysis .................................................................................. 38 2.2.1. Sample preparation from Farfarae Flos ........................................... 38 2.2.2. UHPLC separation ............................................................................. 38 2.2.3. MS/MS analysis ................................................................................... 39 2.2.4. Separation of sesquiterpenoids and structural determination ....... 40 2.2.5. Validation parameters for quantification ......................................... 41 2.3. Activity based proteome profiling: Identification of target proteins of an oplopane sesquiterpenoid in breast cancer cells ..................................... 43 2.3.1. Fractionation of Farfarae Flos extract ............................................. 43 2.3.2. Cell viability assay .............................................................................. 43 2.3.3. Synthesis of ECN-based clickable probe .......................................... 44 2.3.4. Gel-based proteome profiling ............................................................ 45 2.3.5. Preparation of probe-labeled proteome for MS-based analysis ..... 46 2.3.6. LC-MS/MS analysis and data processing ......................................... 48 2.3.7. Modification sites of identified proteins by ECN ............................. 49 2.3.8. Isothermal titration calorimeter ........................................................ 50 IV. RESULTS AND DISCUSSION ..................................................... 52 1. An efficient fractionation method for the preparative separation of sesquiterpenoids from Farfarae Flos by CCC ..................................... 53 1.1. Principle of CCC-DCI fractionation ...................................................... 53 1.2. Selection of the extraction and elution solvents based on KD values ... 55 1.3. CCC-DCI fractionation ........................................................................... 59 1.3.1. Four stages of CCC-DCI .................................................................... 59 1.3.2. Preparative separation of three major sesquiterpenoids ................ 62 1.4. Quantification study ................................................................................ 67 1.4.1. Validation parameters ........................................................................ 68 1.4.2. Comparison of CCC-DCI with conventional methods .................... 70 1.5. Discussion .................................................................................................. 73 2. Chemical profiling of sesquiterpenoids from Farfarae Flos based on LC-MS/MS analysis .................................................................................... 75 2.1. Characterization of diagnostic ions ......................................................... 75 2.1.1. Diagnostic filtering .............................................................................. 78 2.1.2. Fragmentation patterns of the diagnostic ions ................................. 80 2.2. Precursor ion scan for the diagnostic ions .............................................. 82 2.3. Method validation ..................................................................................... 86 2.3.1. Separation of 11 sesquiterpenoids ...................................................... 86 2.3.2. Structural elucidation ......................................................................... 88 2.4. CID-fragmentation behavior of sesquiterpenoids ............................... 105 2.5. Quantification of sesquiterpenoids by MRMHR ................................... 109 2.6. Discussion ................................................................................................ 114 3. Activity-based proteome profiling: Identification of target proteins of an oplopane sesquiterpenoid in breast cancer cells ..................... 116 3.1. Anti-proliferation activities of Farfarae Flos ....................................... 116 3.2. Synthesis of ECN-based clickable probe .............................................. 119 3.3. Gel-based proteome profiling of clickable probe ................................. 125 3.4. MS-based profiling of target proteins of ECN ..................................... 127 3.5. Thermodynamics and binding sites of ECN for target proteins ........ 129 3.6. Discussion ................................................................................................ 133 V. CONCLUSION ..................................................................................... 135 REFERENCES ........................................................................................... 138 ABSTRACT IN KOREAN ................................................................... 157 LIST OF FIGURES Fig. 1. Tussilago farfara L. and Farfarae Flos .............................................. 3 Fig. 2. Chemical structures of reported oplopane and bisabolane type sesquiterpenoids from Farfarae Flos ................................................. 5 Fig. 3. A schematic diagram of CCC system ................................................. 8 Fig. 4. A schematic diagram of CCC separation based on KD value ..... 10 Fig. 5. Polarity correlation between HEMWat systems and isolates ..... 11 Fig. 6. A schematic diagram of LC-MS system .......................................... 13 Fig. 7. A schematic diagram of MS/MS scan modes ................................. 16 Fig. 8. A schematic diagram of LC-MS/MS based dereplication ........... 18 Fig. 9. A schematic diagram of dereplication using MS/MS database ... 19 Fig. 10. A schematic diagram of activity-based proteome profiling ...... 21 Fig. 11. Click chemistry reaction ................................................................... 23 Fig. 12. A schematic diagram of LC-MS based quantitative proteome profiling ................................................................................................ 25 Fig. 13. A schematic diagram of CCC-DCI mode ...................................... 54 Fig. 14. CCC-DCI chromatogram of Farfarae Flos extract .................... 61 Fig. 15. Preparative separation of three major sesquiterpenoids .......... 63 Fig. 16. 1H and 13C spectrum of TG .............................................................. 64 Fig. 17. 1H and 13C spectrum of AECN ........................................................ 65 Fig. 18. 1H and 13C spectrum of ECN ........................................................... 66 Fig. 19. HPLC-UV chromatograms of extract and fraction ................... 71 Fig. 20. In-source fragmentation of sesquiterpenoids ............................... 76 Fig. 21. Proposed diagnostic ions under LC-ESI-MS analysis ............... 77 Fig. 22. Ion chromatogram of STE fraction and diagnostic filtering ..... 79 Fig. 23. Fragmentation patterns of diagnostic ions ................................... 81 Fig. 24. Total ion chromatograms of precursor ion scan ......................... 83 Fig. 25. Preparation of 11 sesquiterpenoids from STE fraction ............. 87 Fig. 26. Precursor ion scans for isolated sesquiterpenoids ...................... 90 Fig. 27. HSQC spectrum of compound No. 7 ............................................. 91 Fig. 28. HSQC spectrum of compound No. 11 ............................................ 92 Fig. 29. HSQC spectrum of compound No. 12 ........................................... 93 Fig. 30. HSQC spectrum of compound No. 14 ........................................... 94 Fig. 31. HSQC spectrum of compound No. 23 ........................................... 95 Fig. 32. HSQC spectrum of compound No. 36 ........................................... 96 Fig. 33. HSQC spectrum of compound No. 39 ........................................... 97 Fig. 34. HSQC spectrum of compound No. 45 ........................................... 98 Fig. 35. HSQC spectrum of compound No. 60 ........................................... 99 Fig. 36. HSQC spectrum of compound No. 68 ......................................... 100 Fig. 37. HSQC spectrum of compound No. 72 ......................................... 101 Fig. 38. NOESY spectrum of compound No. 7 ......................................... 102 Fig. 39. NOESY spectrum of compound No. 23 ....................................... 103 Fig. 40. NOESY spectrum of compound No. 45 ....................................... 104 Fig. 41. Representative MS/MS fragmentation behaviors .................... 107 Fig. 42. Fragmentation behaviors of mono- and hetero-isotopic ions ... 108 Fig. 43. Herbal materials for quantification study .................................. 110 Fig. 44. Dereplication of 8 sesquiterpenoids by UHPLC-MRMHR ...... 111 Fig. 45. Anti-proliferation activities of fractions from Farfarae Flos .. 117 Fig. 46. Anti-proliferation activities of compounds from Farfarae Flos 118 Fig. 47. Synthesis of ECN-based clickable probe and anti-proliferation activity ................................................................................................. 120 Fig. 48. HSQC spectrum of ECN ................................................................. 121 Fig. 49. HSQC spectrum of ECN-E ............................................................ 122 Fig. 50. HSQC spectrum of ECN-N3 ........................................................... 123 Fig. 51. 15N-HMBC spectrum of ECN-N3 .................................................. 124 Fig. 52. Gel-based profiling of ECN-N3 labeled proteome in situ ........ 126 Fig. 53. Thermograms and parameters for interaction of ECN with identified target proteins ................................................................ 131 Fig. 54. Alkylation of cysteine residues in 14-3-3 protein zeta by ECN .. 132 Fig. 55. Alkylation of cysteine residues in peroxiredoxin-1 by ECN ..... 133 LIST OF TABLES Table 1. The partition coefficients (KD) of three major sesquiterpenoids in different solvent composition ..................................................... 57 Table 2. Comparison of the extraction efficiency of 45% acetonitrile and methanol .............................................................................................. 58 Table 3. The linear range, linearity, LOD, and LOQ of three major sesquiterpenoids by UV detection ................................................. 69 Table 4. The comparison of the fractionation efficiency of CCC-DCI, solvent partitioning, and open column chromatography ........ 72 Table 5. Identified sesquiterpenoids of Farfarae Flos by precursor ion scan of UHPLC-QqQ-MS/MS ........................................................ 84 Table 6. Quantitative parameters for sesquiterpenoids by UHPLC- MRMHR .............................................................................................. 112 Table 7. Intra-day and inter-day precision of UHPLC-MRMHR ......... 112 Table 8. Extraction yield of herbal materials ........................................... 113 Table 9. Contents of 8 sesquiterpenoids in Tussilago farfara by UHPLC- MRMHR .............................................................................................. 113 Table 10. Identified target proteins of ECN in breast cancer cells ...... 129 | - |
| dc.language.iso | eng | - |
| dc.publisher | 서울대학교 대학원 | - |
| dc.subject | 관동화 | - |
| dc.subject | 세스퀴테르펜 | - |
| dc.subject | 향류크로마토그래피 | - |
| dc.subject | 직접연속주입법 | - |
| dc.subject | 질량분석기반 성분프로파일 | - |
| dc.subject | 세포증식억제 | - |
| dc.subject | 생리활성기반 표적단백체 | - |
| dc.subject.ddc | 615 | - |
| dc.title | Chemical profiling of Farfarae Flos sesquiterpenoids and the target protein identification of an oplopane sesquiterpenoid in breast cancer cells | - |
| dc.title.alternative | 관동화 유래 세스퀴테르펜 화합물의 성분프로파일 및 유방암 세포주에서의 oplopane 세스퀴테르펜의 표적 단백질에 관한 연구 | - |
| dc.type | Thesis | - |
| dc.type | Dissertation | - |
| dc.contributor.AlternativeAuthor | Song, Kwangho | - |
| dc.contributor.department | 약학대학 약학과 | - |
| dc.description.degree | Doctor | - |
| dc.date.awarded | 2019-08 | - |
| dc.contributor.major | 천연물과학 | - |
| dc.identifier.uci | I804:11032-000000156811 | - |
| dc.identifier.holdings | 000000000040▲000000000041▲000000156811▲ | - |
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