초록 ▼

본 연구에서는 1) 전통천연물의 MC/MT 독성 해석 및 부작용올 완화시키고 효능을 극대화할 수 있는 소재 도출을 위해 combinatorial optimization 기반 독성후보물질의 상호작용올 분석하고 2) 독성 예측모델 개발을 위한 기초자료로 활용될 수 있도록 생산된 데이터를 database화하고 동물 모델을 이용하여 microarray 분석를 통한 독성 바이오마커의 후보를 발굴하고자 하였다. 먼저 combinatorial optimization 기반 독성후보물질의 상호작용을 분석한 결과， 태음조위탕 구성 천연물 가운데 천문

본 연구에서는 1) 전통천연물의 MC/MT 독성 해석 및 부작용올 완화시키고 효능을 극대화할 수 있는 소재 도출을 위해 combinatorial optimization 기반 독성후보물질의 상호작용올 분석하고 2) 독성 예측모델 개발을 위한 기초자료로 활용될 수 있도록 생산된 데이터를 database화하고 동물 모델을 이용하여 microarray 분석를 통한 독성 바이오마커의 후보를 발굴하고자 하였다. 먼저 combinatorial optimization 기반 독성후보물질의 상호작용을 분석한 결과， 태음조위탕 구성 천연물 가운데 천문동의 독성을 완화시키는 천연물로는 석창포， 의이인이， 마황은 농도에 따라 길경 또는 맥문동 등 천연물 조합에 따라 다른 효과를 보였으며， 길경의 독성 완화에 관여하는 천연물은 내복자와 맥문동으로 분석되었다. 녹차 카테킨류 최적화 실험올 통해 독성을 낮추고 활성을 높이는 인자로 ( -) - Epigallocatechin gallate(EGCG) 과 ( + ) -Gallocatechin 이 도출되었으며， 도출된 상호작용을 검중한 결과 EGCG와 ( + ) -Gallocatechin을 동시 처리시 ( + ) - Gallocatechin에 의해 유도되는 세포독성은 감소되었으며， 염중관련 NO 생성의 저해효과는 유의적으로 증대됨을 확인하고 관련 데이터들은 database화 하였다. 전통천연물의 세포독성 기전을 이해하고자 태음조위탕 구성 천연물이 ER stress 유발에 미치는 영향을 살펴본 결과， 태음조위탕 구성 천연물 가운데 의이인 주정 추출물이 ER stress marker 인 CHOP/Gadd153, BIP/GRP78 의 유전자 및 단백질 발현 증가와 XRP1 splicing 이 관찰되어 ER stress를 유발할 가능성이 있는 것으로 보였다. 14-day 마우스 반복 경구 독성 모댈에서 태음조위탕 및 내복자 추출물 투여에 의해 간 또는 신장 조직에 대해 mlcroarray 분석을 실시한 결과， 내복자 주정 추출물을 투여한 신장조직의 유의적인 유전자 발현 차이가 뚜렷하였다. 이에 급성신장 injury와 관련하여 보고된 임상 바이오마커와 본 연구를 비교한 결과 kim-1 , S100a8, Lcn2 등이 내복자 고용량 투여군에서 증가하는 것으로 나타났으며 ， 특히 kim-l 의 경우 내복자 투여 신장 조직을 대상으로 살펴본 결과 female 10 g/kg 내복자투여군에서 발현 증가가 확인되었다. 이외에도 추가적인 데이터 분석올 통해 독성 바이오마커 후보를 추가적으로 발굴해 나갈 예정이다.

(출처:보고서 요약서 p.5)

Abstract ▼

IV. Results

A. Screening of multicomponent natural materials for studying toxicity biomarkers
○ In order to evaluate toxic interactions of multi -components and/or natural products and screen new toxicity biomarkers, Tae-Um-Jo-Wee-Tang, green tea, Coicis semen, Raphani semen and their main

IV. Results

A. Screening of multicomponent natural materials for studying toxicity biomarkers
○ In order to evaluate toxic interactions of multi -components and/or natural products and screen new toxicity biomarkers, Tae-Um-Jo-Wee-Tang, green tea, Coicis semen, Raphani semen and their main constituents were selected and examined.

B. Screening the optimal data mining methods
○ A variety of natural products and/or their constituents are possible to possess both toxicity and beneficial effects in a complex way. To extract information from a data set of toxicity on natural products and/or their constituents and transform it into an interactions of natural products and/or their constituents, data mmmg methods such as robust data mining procesure (RDMP) , correlation based feature selection (CBFS) and best first search (BFS) were used in this study.

C. Combinatorial optimization
○ Optimized combinations of green tea catechins and eight medicinal plants consisting of Tae-Um-Jo-Wee-Tang and for minimized cytotoxicity and maximized bioactivity (eg.anti - inflammatory activity) were drawn based on combinatorial optimization.

○ Customized statistical design was applied to investigate interactions between eight medicinal plants consisting of Tae-Um-Jo-Wee-Tang, and draw the optimal combination exerting minimized cytotoxicity. As a result , Asparagi Tuber, Platycodi Radix and Ephedrae Herba showed more potent cytotoxicity than others as tested by lactate dehydrogenase (LDH) assay. A number of combinations at several concentrations were identified and re-confirmed with detailed combination.

○ Customized statistical design was applied to investigate interactions between green tea catechins (Epigallocatechin gallate (EGCG) , Epigallocatechin (EGC) , Epicatechin gallate (ECG) , Epicatechin (EC) , Gallocatechin and Catechin) , and draw the optimal combination exerting minimized cytotoxicity and maximized nitric oxide (NO) inhibitory activity. The cytotoxicity of catechins against liver cells was tested using lactate dehydrogenase (LDH) assay and the inhibitory effect on nitric oxide (NO) production was determined in lipopolysaccharide-induced macrophage. According to the experimental design, it was confirmed with 28 combination at two concentrations, re-confirmed with detailed combination, and finally validated by LDH and NO assays.

D. Interactions of MC natural products
○ Among eight medicinal plants consisting of Tae-Um-Jo-Wee-Tang, cytotoxicity induced by Asparagi Tuber was reduced by Coicis semen and/or Acori Gramineri Rhizoma. In contrast, cytotoxicity induced by Platycodi Radix was reduced by Raphani semen and/or Liriopis Tuber.

○ Among Green tea catechins, EGCG and Gallocatechin combination out of 6 catechins significantly enhanced NO inhibitory activity and reduced cytotoxicity. To our best knowledge, it is the first report providing the platform for finding the optimized combination of food components exerting enhanced efficacy and reduced toxicity at the same time.

E. Toxicity of MC natural products on the cell organelles
○ Endoplasmic reticulum stress (ER stress) is key process to induce various diseases such as inflammation, type 2 diabetes and Alzheimer’s disease. Coicis semen (Adlay seed, Job’s tears) has been widely consumed as a herbal medicine in South Korea.
○ It was investigated whether Coicis semen and coixol (6-methoxy-l ,3-benzoxazol-2(3H)-one) induce ER stress in Chang liver cell. The expression of ER stress marker including binding immunoglobulin protein (BiP) , CÆBP homologous protein (CHOP) and X-box binding protein 1(XBP1) measured by qRT-PCR, XBPl splicing assay and Western blot analysis. Coicis semen ethanol extracts (CSE) and coixol inhibited cell proliferation with IC50 as 250 and 350. Similarly, CSE treatment with 400 and 500 / increased spliced XBP1. However, coixol has not affected splicing of XBPl. These results suggest that Coicis semen on high dose has a potential of induced ER stress on liver cells.

F. Database for interactions of MC natural products
○ The database was established by using ‘ACCESS’ program for cytotoxicity data (1 ,245 data for green tea catechins, 168 data for eight medicinal plants consisting of Tae-Um-Jo-Wee-Tang) conducted in this study. Additional data will be added continuously.

G. In vivo oral toxicity and Screening for the toxicity biomarkers
○ In order to find acute and early toxicity biomarkers, 14-day repeated dose toxicity study in mouse was performed. Each group (n=10, five rriale and five female mice) were administered orally with extracts (Tae-Um-Jo-Wee-Tang and Raphani semen) at the dose of 0 (1% CMC) -10 g/kg bw daily once for a period of 14 days to obtain the signatures of toxicity. After two weeks, haematological, biochemical and histological examinations were performed. More importantly, mouse liver and/or kidney were subject to microarray analysis.

○ Hot water extract of Tae-Um-Jo-Wee-Tang altered the expression of 217 and 208 genes for female and male, respectively, after 14-day of exposure. Of particular interest, transcripts encoding cytochrome P450, infiammation, lipid metabolism, and MAPK signal transduction were upor down-regulated by hot water extract of Tae-Um-Jo-Wee-Tang. The color scale expressed as heat map reflects the experimental fold increase (red) or fold decrease (green) in transcript abundance relative to the corresponding control experiment.

○ For the DNA microarray experiments of Raphani semen, array data was filtered, normalized and analyzed. Then gene set enrichment analysis (GSEA) was carried out to select the significant gene sets which were significantly affected by this herbal extract. Significant gene set in mouse liver was analyzed to be ‘cholesterol biosynthesis genes'. In addition, significant biological process based on the gene ontology affected by the herbal extract or their components was found to be ‘inflammatory re sponse' and ‘acute-phase response'.

○ The GSEA results for mouse kidney showed that the most significant gene sets in response to this extract among 74 gene sets were ‘DNA replication' and ‘cell cycle'. In addition, significant biological process based on the gene ontology affected by the herbal extract was found to be ‘inflammatory response' and ‘acute-phase response '. From significant genes, over 10 known genes per each extract were selected and is validating using realtime PCR.

○ DNA microarray data from the extracts of Tae-Um-Jo-Wee-Tang and Raphani semen were provided to the database team to create the database for the biomarker genes of herbal medicine or functional foods.

H. Selection of the toxicity biomarker candidates
○ A comparison of gene expression profiles between liver and kidney indicates that gene expression in the liver was less affected by extract from Raphani semen than that in the kidney. The most a ffected gene sets in response to extract from Raphani semen in the kidney included DNA replication, cell cycle, infiammation, apoptosis etc., many of which are relevant to adverse effect of Raphani semen at high dose. Among significant genes affected by extract of Raphani semen in the kidney, several genes in cluding Kim-l , SlOOa8 and Lcn2 are thought to be a potential toxicity biomarker candidates respons ible for Raphani semen -mediated changes. Further, new candidate genes will be exploited by the additional data analysis.

(출처:SUMMARY p.13)

목차 Contents

• 표지 ... 1
• 제출문 ... 3
• 보고서 요약서 ... 5
• 요약문 ... 7
• SUMMARY ... 13
• CONTENTS ... 17
• 목차 ... 19
• 제 1 장 연구개발과제의 개요 ... 21
• 1절. 연구개발의 필요성 ... 21
• 2절. 연구개발의 목적(당해 단계) ... 22
• 3절. 연구개발의 내용 및 범위 ... 22
• 제 2 장 국내외 기술개발 현황 ... 23
• 1절. 국내 연구동향 ... 23
• 2절. 국외 연구동향 ... 24
• 제 3 장 연구개발수행 내용 및 결과 ... 28
• 1절. 독성 후보 물질 탐색 ... 28
• 2절. 적정 data mining 분석법 탐색 ... 28
• 3절. Combinatorial optimization 분석 및 독성 후보물질의 상호작용 분석 ... 28
• 4절. 독성후보 물질의 상호작용 database 구축 ... 57
• 5절. 세포 소기관 독성 여부 분석 ... 59
• 6절. 독성 유발 조건 확립 및 독성 바이오마커 스크리닝 ... 74
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 97
• 1절. 연구목표 달성도 ... 97
• 2절. 관련 분야에의 기여도 ... 99
• 3절. 연구개발의 최종목표 ... 100
• 제 5 장 연구개발결과의 활용계획 ... 103
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 104
• 제 7 장 참고문헌 ... 107
• 끝페이지 ... 110