Angular Dihydropyranocoumarins from Peucedanum japonicum Roots

학위논문 (박사)-- 서울대학교 대학원 약학대학 약학과, 2017. 8. 김진웅.Peucedanum japonicum Thunberg, belongs to Umbelliferae family, was distributed in southern and eastern Asian countries. Its roots were traditionally used as a medicine for cold and neuralgic diseases in Korea and Taiwan. It was reported that the roots of this plant contained coumarins, chromones, polyacetylenes, sugar alcohols, and steroid glycosides. Pharmacological researches revealed that P. japonicum roots showed antioxidative, anti-inflammatory, antifungal activity and cytotoxic effect against lymphocytic leukaemia. In this study, sixteen new angular dihydropyranocoumarins (1-16) and three new angular monohydromonohydroxyfuranocoumarins (41-43) were isolated along with forty-five known compounds from the n-hexane and CHCl3 fractions of the P. japonicum roots. The known compounds were characterized as angular dihydropyranocoumarins (17-40), linear furanocoumarins (44-47), an angular dihydrofuranocoumarin (48), a linear dihydropyranocoumarin (49), simple coumarins (50-58), a chromone (59), ferulic acid derivatives (60-61), a lignan (62), a phenylpropanoid (63), and an indole alkaloid (64). Isolated angular dihydropyranocoumarins (khellactones) included monoacyl- and diacyl-type khellactone esters. In the case of monoacylkhellactones, the absolute configuration was easily determined by the Mosher method. However, the absolute configuration of diacyl khellactone esters was difficult to assign due to the absence of free hydroxyl group. Therefore, partial alkaline hydrolysis prior to MTPA derivatization and X-ray diffraction analysis were applied to determine the absolute configurations at 3- and 4-positions. Because the success of partial hydrolysis and single crystallization was very difficult, ECD spectroscopy was suggested to confirm the absolute configuration of most of the compounds. Interestingly, a few enantiomers were discovered and isolated by enantio-selective column. Enantiomers were usually detected and isolated using chiral column. However, RP-HPLC analysis with MTPA reaction products could be alternative method to confirm enantiomer existence without testing a number of chiral-selective columns. In the case of cis-monoacylkhellactones, the interconversion of substituents at 3- and 4-position was observed. The major MS fragment peak of khellactone esters was detected without a C-4 substituent. Thus, the position of substituents at 3 and 4 could be determined by MS fragmentation analysis without HMBC measurement. The NO production inhibitory activity of isolated compounds were tested using Griess assay in LPS-induced RAW264.7 cells. As a result, 1, 3-6, 22, 31, and 36-38 showed significant activity without cytotoxicity. The isobutyryl, senecioyl, 2-methylbutyryl, and isovaleryl moieties at 3 position played more important role than the acetyl and angeloyl groups. In conclusion, the absolute configuration of isolated compounds were assigned by various methods. Those methods will become useful guide to solve similar structures. The isolated compounds which significantly inhibited NO production were suggested as anti-inflammatory candidates from natural resources.Chapter 1. Introduction 1 1.1. Study Background 1 1.1.1. The genus Peucedanum and the species Peucedanum japonicum Thunberg 1 1.1.2. Angular dihydropyranocoumarins 4 1.1.3. Inflammation and the role of nitric oxide 4 1.2. Purpose of research 5 Chapter 2. Structure Elucidation of Khellactone Esters 6 2.1. Compound 1 6 2.2. Compound 2 10 2.3. Compound 9 13 2.4. Compounds 8, 10, 11, and 15 15 2.5. Compound 6 19 2.6. Compounds 3, 4, 7, and 13 20 2.7. Compounds 5, 21, 22, and 23 23 2.8. Compounds 12, 24, 25, 26, 27, and 40 26 2.9. Compounds 14, 28, 29, and 30 31 2.10. Compounds 31, 32, 33, and 34 34 2.11. Compounds 35, 36, and 37 37 2.12. Compounds 38 and 39 39 2.13. Compounds 16, 17, 18, 19, and 20 41 Chapter 3. Structure Elucidation of Angular Furanocoumarins 48 3.1. Compounds 41, 42 and 43 48 3.2. Compound 48 54 Chapter 4. Structure Elucidation of Other Compounds 55 4.1. Compound 44 55 4.2. Compounds 45, 46, and 47 56 4.3. Compound 49 58 4.4. Compounds 50 and 51 59 4.5. Compounds 52, 53, 54, and 55 61 4.6. Compounds 56, 57, and 58 64 4.7. Compound 59 66 4.8. Compounds 60 and 61 67 4.9. Compound 62 69 4.10. Compound 63 70 4.11. Compound 64 71 Chapter 5. Remark 74 5.1. Acyl migration 74 5.2. MS fragmentation 76 Chapter 6. Bioactivity of the Isolated Compounds 78 6.1. NO production inhibitory activity of isolated compounds 78 Chapter 7. Experimental Section 79 7.1. Materials 79 7.1.1. Plant material. 79 7.1.2. Reagents 79 7.1.3. Equipments 79 7.2. Extraction and fractionation of P. japonicum 81 7.3. Isolation of compounds from n-hexane and CHCl3 fractions 82 7.4. Spectroscopic and spectrometric data of isolated compounds 87 7.4.1. (3S,4S)-3'-O-isobutyryl-4'-O-(2-methylbutyryl)khellactone (1) 87 7.4.2. (3S,4S)-3'-O-acetyl-4'-O-senecioylkhellactone (2). 87 7.4.3. (3S,4S)-4'-O-isobutyryl-3'-O-(2-methylbutyryl)khellactone (3) 87 7.4.4. (3S,4S)-3'-O-(2-methylbutyryl)-4'-O-senecioylkhellactone (4) 88 7.4.5. (3S,4S)-4'-O-(2-methylbutyryl)-3'-O-senecioylkhellactone (5) 88 7.4.6. (3S,4S)-3'-O-isobutyryl-4'-O-isovalerylkhellactone (6) 89 7.4.7. (3S,4S)-4'-O-angeloyl-3'-O-(2-methylbutyryl)khellactone (7) 89 7.4.8. (3S,4S)-3'-O-butyryl-4'-O-(2-methylbutyryl)khellactone (8). 89 7.4.9. (3S,4S)-4'-O-angeloyl-3'-O-isovalerylkhellactone (9) 90 7.4.10. (3S,4S)-3'-O-acetyl-4'-O-(3-hydroxyisovaleryl)khellactone (10) 90 7.4.11. (3S,4S)-3'-O-acetyl-4'-O-(3-hydroxy-2-methylbutyryl)khellactone (11) 90 7.4.12. (3S,4S)-3'-O-acetyl-4'-O-(2-methylbutyryl)khellactone (12) 91 7.4.13. (3S,4S)-3'-O-(2-methylbutyryl)khellactone (13) 91 7.4.14. (3S,4S)-4'-O-(2-methylbutyryl)khellactone (14) 91 7.4.15. (3S,4S)-4'-O-methyl-3'-O-(2-methylbutyryl)khellactone (15) 92 7.4.16. (3S,4R)-4'-O-senecioylkhellactone (16) 92 7.4.17. (3S,4S)-4'-O-senecioylkhellactone (17) 92 7.4.18. (3R,4R)-4'-O-senecioylkhellactone (18) 93 7.4.19. (3S,4S)-3'-O-senecioylkhellactone (19) 93 7.4.20. (3R,4R)-3'-O-senecioylkhellactone (20) 93 7.4.21. (3S,4S)- 4'-O-angeloyl-3'-O-senecioylkhellactone (21) 94 7.4.22. (3S,4S)- 3',4'-di-O-senecioylkhellactone (22) 94 7.4.23. (3S,4S)-4'-O-isovaleryl-3'-O-senecioylkhellactone (23) 95 7.4.24. (3S,4S)-3'-O-acetylkhellactone (24) 95 7.4.25. (3S,4S)- 3-O-acetyl-4-O-angeloylkhellactone (25) 95 7.4.26. (3S,4S)-3'-O-acetyl-4'-O-isobutyrylkhellactone (26) 96 7.4.27. (3S,4S)-3'-O-acetyl-3'-O-isovalerylkhellactone (27) 96 7.4.28. (-)-cis-khellactone (28) 96 7.4.29. (3S,4S)-4'-O-acetylkhellactone (29) 97 7.4.30. (3S,4S)-3-hydroxy-4-O-angeloyloxy-3,4-dihydroseelin (30) 97 7.4.31. (3S,4S)-3'-O-angeloyl-4'-O-(2-methylbutyryl)khellactone (31) 97 7.4.32. (3S,4S)-3',4'-di-O-angeloylkhellactone (32) 98 7.4.33. (3S,4S)-3-O-angeloyloxy-4-hydroxy-3,4-dihydroseselin (33) 98 7.4.34. (3S,4S)-3-O-angeloyl-4-O-senecioylkhellactone (34) 98 7.4.35. (3S,4S)-3'-O-isovaleryl-4'-O-senecioylkhellactone (35) 99 7.4.36. (3S,4S)-3',4'-di-O-isovalerylkhellactone (36) 99 7.4.37. (3S,4S)-3'-O-isovaleryl-4'-O-(2-methylbutyryl)khellactone (37) 99 7.4.38. (3R)-O-senecioyllomatin (38) 100 7.4.39. (3R)-O-isovaleroyllomatin (39) 100 7.4.40. (3R,4R)-3,4-di-O-acetylkhellactone (40) 100 7.4.41. 2-hydroxy-3-O-senecioylvaginol (41) 101 7.4.42. 2-hydroxy-3-O-(2-methylbutyryl)vaginol (42) 101 7.4.43. 2-hydroxy-3-O-isovalerylvaginol (43) 101 7.4.44. (+)-marmesin (nodakenetin) (44) 102 7.4.45. 9-(2-hydroxy-3-methoxy-3-methylbutoxy)bergapten (45) 102 7.4.46. isoimperatorin (46) 103 7.4.47. 5-(2-hydroxy-3-methoxy-3-methylbutoxy)psoralen (47) 103 7.4.48. 3-O-senecioylvaginidiol (48) 104 7.4.49. (S)-(+)-decursin (49) 104 7.4.50. isoarnottinin (50) 105 7.4.51. umbelliferone (51) 105 7.4.52. scoparone (52) 106 7.4.53. tamarin (isosuberenol) (53) 106 7.4.54. (Z)-suberenol (54) 106 7.4.55. suberosin (55) 107 7.4.56. peucedanol (56) 107 7.4.57. peucedanol 7-O-β-D-glucopyranoside (57) 108 7.4.58. peujaponiside (58) 108 7.4.59. eugenin (59) 109 7.4.60. 6,β-dihydroxyphenethyl trans-ferulate (decursidate) (60) 109 7.4.61. 6-hydroxyphenethyl cis-ferulate (61) 110 7.4.62. (-)-pinoresinol (62) 110 7.4.63. trans-ferulic acid (63) 111 7.4.64. 3-formylindole (64) 111 7.5. Partial and total alkaline hydrolysis of 1 123 7.6. Preparation of MTPA esters of 1a 123 7.7. Preparation of MTPA esters of 16 124 7.8. Preparation of MTPA esters of 17 and 18 125 7.9. Preparation of MTPA esters of 19 and 20 125 7.10. X-ray crystallographic analysis of 1 and 2 127 7.10.1. Crystal data of 1 127 7.10.2. Crystal data of 2 127 7.11. ECD calculation 128 7.12. Evaluation of inhibitory effect on NO production in LPS-stimulated RAW 264.7 cells 129 7.12.1. Reagents 129 7.12.2. Cell cultures 129 7.12.3. Griess assay 129 7.12.4. MTT assay 130 Chapter 8. Conclusion 131 References 133 국문초록 140Docto

백출의 대식세포 내 산화질소 생성 저해 성분

학위논문 (석사)-- 서울대학교 대학원 : 약학과, 2012. 2. 김진웅.본 연구에서는 염증성 질환을 예방하거나 치료할 수 있는 후보물질을 천연물로부터 도출하기 위하여 lipopolysaccharide (LPS)로 염증반응을 유도한 male mouse 유래 macrophage RAW264.7 세포주를 검색계로 하였다. 천연물을 대상으로 nitric oxide (NO) 생성 억제 활성을 검색하던 중 삽주 뿌리줄기의 총 메탄올 추출물이 유의성 있는 억제 활성을 나타냄을 확인하였다. 백출(Atractylodis Rhizoma Alba)은 삽주의 뿌리줄기 또는 주피를 제거한 것이다. 삽주 (Atractylodes japonica Koidz.)는 국화과(Compositae)에 속하는 식물로서 뿌리줄기는 방향성 건위제로 진정, 이뇨, 지한, 자양, 안태, 면역증강, 항미생물, 항염증을 목적으로 사용되어왔다. 백출은 sesquiterpene과 그 배당체 계열 성분과 polyacetylene 계열 성분 등이 주성분으로 보고되어 있고, 항염 활성에 대하여 보고된 적이 있으나, 이와 관련된 성분 연구는 아직까지 체계적으로 이루어진 바 없다. 이에 본 연구에서는 항염 활성을 갖는 성분을 활성지향적인 방법으로 분리, 규명한 후, Griess assay를 이용하여 산화질소 생성 억제능을 검토하고자 하였다. 건조된 백출 약 4 kg의 100% MeOH 추출물을 증류수에 현탁한 후 용매의 극성에 따라 n-hexane, CHCl3, EtOAc, n-BuOH, 수용액 층으로 각각 분획하였다. 이 중 유의성 있는 NO 생성 억제 활성을 나타낸 n-hexane, CHCl3 분획물을 대상으로 silica gel, sephadex LH-20 column chromatography와 HPLC 등을 차례로 진행하여 활성지향적 분리기법으로 총 14종의 화합물을 분리하였다. 각각의 화합물들은 UV, IR, CI/ESI-MS, 1H-NMR, 13C-NMR, 1H-1H COSY, selective TOCSY, HMQC, HMBC, NOESY, DEPT spectrum과 선광도 등의 분광학적 데이터와 이화학적 성상을 종합하여 atractylenolide III (1), atractylenolide II (2), 8β-methoxyatractylenolide (3), 4,15-epoxy-8β-hydroxyasterolide (4), (3R)-3-hydroxyatractylenolide III (5), taenialactam B (6), 8-epiasterolide (7), eudesma-3,7(11)-dien-5,15-dihydroxy-8α,13-olide (8), 4(15)-eudesmene-1β,7,11-triol (9), 3-eudesmene-1β,7,11-triol (10), eudesm-7(11)-en-4α-ol (11), eudesm-4(15),7-diene-11-ol (12), atractylodiol (13), daucosterol (14)로 동정하였다. 화합물 1～12는 sesquiterpenene 계열, 13은 polyacetylene 계열, 14는 steroid 배당체 계열 물질이다. 이들 화합물 중 8, 12는 천연에서 처음 분리, 보고되는 물질이고, 화합물 5, 6, 9, 10은 이 과 식물에서, 화합물 7은 이 속 식물에서, 화합물 3, 4, 11은 이 식물에서 처음 분리, 보고되는 물질이다. RAW264.7 세포주를 이용한 Griess assay를 시행하여 분리된 화합물들의 산화질소 생성 억제 활성을 측정하였다. 화합물 중 2, 10이 용량 의존적으로 산화질소의 생성을 억제하는 것으로 나타났으며 8-hydroxyl moiety가 없는 화합물 2가 화합물 1, 4, 5보다 유의성있게 억제활성을 나타내었다.Maste

Airborne precautions based on Xpert ® MTB/RIF results for patients with presumptive TB

Airborne precautions based on Xpert ® MTB/RIF results for patients with presumptive T

Comparison of the Microbiological Efficacy of Disinfection Using Ultraviolet and Hydrogen Peroxide System for Carbapenemase-producing Enterobacteriaceae in a Healthcare Setting

Background: This study aimed to compare the efficacy of microbiological disinfection between the ultraviolet-C (UV-C) device and aerosolized hydrogen peroxide (aHP) system in a healthcare setting. Methods: Four rooms were installed with two UV-C devices and two aHP systems. Thirty formica sheets contaminated with carbapenemase-producing Enterobacteriaceae (CPE) were placed in each room. After intervention, the median log10 reduction and modified decontamination rates were compared between the two methods using Rodac plates. Eight sink drains in the rooms previously occupied by a patient with CPE were sampled separately before and after the interventions. Results: The median log10 reduction was 5.52 and 5.37 after the UV-C (n=60) and aHP (n=60) interventions, respectively (P=0.86), whereas the modified decontamination rate was 50% and 45%, respectively (P=0.71). At the UV-direct sites, UV-C showed higher median log10 reduction (5.91 vs. 5.61, P=0.002) and modified decontamination rate (83% vs. 53%, P=0.03) than those of aHP. Conversely, at UV-indirect sites, aHP showed higher median log10 reduction (4.63 vs. 5.07%, P=0.02) and modified decontamination rate (17% vs. 37%, P=0.01) than those of UV-C. After the intervention, carbapenemase-resistant Gram-negative bacilli decreased further in five of the seven sink drains disinfected by sodium. Conclusion: Both UV-C and aHP reduced the bacterial contamination in the rooms. The aHP was significantly more effective than UV-C at the UV-indirect sites, and the converse was true for the UV-direct sites. Application of the intervention to disinfect the sink drains resulted in additional bacterial decontamination. Considering the features of the machines and the results of this study, healthcare facilities can choose either UV-C or aHP for decontamination

Frequency of and risk factors for reversion of QuantiFERON test in healthcare workers in an intermediate-tuberculosis-burden country

Objectives: High-risk healthcare workers (HCWs) are often screened for latent tuberculosis infection (LTBI) using QuantiFERON tests (QFTs), with annual serial tests often showing reversion from positive to negative results. We assessed the frequency of and risk factors for reversion of QFTs in HCWs in an intermediate-tuberculosis burden country. Methods: We enrolled high-risk HCWs at a tertiary-care hospital in South Korea, who were assessed by QFTs at least twice between 2017 and 2019. Results: Of the 1870 HCWs screened, 1542 (82%) had persistent negative results, 229 (12%) had persistent positive results, 53 (3%) showed reversion, and 46 (2%) showed conversion from negative to positive. Multivariate analysis comparing the characteristics of the 229 HCWs with persistent positive results and the 53 who experienced reversion showed that older age (adjusted odds ratio (aOR): 0.96; 95% confi-dence interval (CI): 0.92-0.99), male sex (aOR: 0.29; 95% CI: 0.11-0.78) and high (>0.70 IU/mL) baseline QFT results (aOR: 0.15; 95% CI: 0.07-0.31) were inversely associated with reversion. Using an ROC curve derived cut-off of <0.738 IU/mL, the area under the curve was 0.79. Of 53 HCWs with reversion, 36 (78%) had below 0.738 IU/mL of baseline QFT, while 181 (79%) of 229 HCWs without reversion had above 0.738 IU/mL of baseline QFT. Conclusion: Reversion during serial testing is unlikely in HCWs who are male, older in age, and have higher baseline QFT results. Serial testing without LTBI treatment may be indicated in HCWs who are female, younger and, especially, have lower QFT results. Ja Young Kim, Clin Microbiol Infect 2021;27:1120 (c) 2020 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved

Nosocomial Outbreak of COVID-19 in a Hematologic Ward

Background Coronavirus disease 2019 (COVID-19) outbreaks occur in hospitals in many parts of the world. In hospital settings, the possibility of airborne transmission needs to be investigated thoroughly. Materials and Methods There was a nosocomial outbreak of COVID-19 in a hematologic ward in a tertiary hospital, Seoul, Korea. We found 11 patients and guardians with COVID-19 through vigorous contact tracing and closed-circuit television monitoring. We found one patient who probably had acquired COVID-19 through airborne-transmission. We performed airflow investigation with simulation software, whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Results Of the nine individuals with COVID-19 who had been in the hematologic ward, six stayed in one multi-patient room (Room 36), and other three stayed in different rooms (Room 1, 34, 35). Guardian in room 35 was close contact to cases in room 36, and patient in room 34 used the shared bathroom for teeth brushing 40 minutes after index used. Airflow simulation revealed that air was spread from the bathroom to the adjacent room 1 while patient in room 1 did not used the shared bathroom. Airflow was associated with poor ventilation in shared bathroom due to dysfunctioning air-exhaust, grill on the door of shared bathroom and the unintended negative pressure of adjacent room. Conclusion Transmission of SARS-CoV-2 in the hematologic ward occurred rapidly in the multi-patient room and shared bathroom settings. In addition, there was a case of possible airborne transmission due to unexpected airflow