[보고서]장내미생물에 의한 독성 평가기술 개발

장내미생물에 의한 독성 평가기술 개발
Development of toxicity tesing methods using intestinal microflora 원문보기

보고서 정보
주관연구기관	영남대학교 YeungNam University
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2011-11
과제시작연도	2011
주관부처	식품의약품안전청 Korea Food & Drug Administration
등록번호	TRKO201600010664
과제고유번호	1475006449
사업명	안전성관리기반연구
DB 구축일자	2016-10-29
키워드	장내미생물총.독성평가.대사효소.시험관내시험.생체시험.intestinal microflora.toxicity testing.fecalase.in vitro test.in vivo test.
DOI	https://doi.org/10.23000/TRKO201600010664

초록 ▼

○ 연구목표
◦ 장내미생물 배양법을 이용한 미생물 대사체 독성 평가시험법의 개발
◦ 한국인(50인 이상) 분변미생물의 합성기질, 천연성분의 대사활성 분석
◦ 합성기질, 천연물성분(의약품 포함)에 대한 fecalase의 기질 특이성, 한국인 성인 표준 fecalase 대사활성 설정 및 표준 대사활성을 갖는 fecalase 제조방법에 관한 SOP 작성
◦ 장내미생물을 이용한 in vivo 대사독성 평가모델 정립
◦ 합성기질 및 천연성분의 대사활성에 대한 기질 특이성을 분석하여 fecalase를 대신할 수 있는 약물대사효소계(intestinal microbial enzyme mix)의 제조

○ 연구내용
장내미생물 배양법을 이용한 미생물 대사체 독성 평가시험법 개발
- 장내미생물 배양기술 확립 및 대사체 분석법 개발
- Fecalase 및 intestinal microbial enzyme mix를 이용한 대사체 독성평가 기술개발
- 장내미생물 대사체의 면역독성 평가
- 장내 미생물을 이용한 세포독성 및 세포사멸 평가기술 개발
- 장내 미생물을 이용한 유전독성 평가기술 개발

장내세균총 유발 독성평가를 위한 장내세균효소 복합체(fecalase) 개발
- 한국인 50인 이상 (총 150인 이상)의 분변 미생물의 합성기질, 천연성분, 천연추출물의 대사활성을 분석하여 한국인 평균 장내세균총의 약물대사활성 도출
- 한국인 성인 표준 fecalase 대사활성 설정
- 한국인의 소화관으로부터 분리한 intestinal microbial enzyme mix의 제조
- 약물 및 독성물질 대사에 있어 장내미생물 대사의 젓요성 평가
- Fecalase 및 intestinal microbial enzyme mix의 제조방법에 관한 표준작업수순서 작성

In vivo 미생물 대사체 독성 평가기술 개발
- 무균동물의 확보, 유지 및 공급시스템의 확립
- 장내미생물 정착균동물의 확립
- 무균동물 및 장내미생물 정착균동물을 활용한 동태연구
- 항생제 처치동물에서의 식품 또는 의약품의 동태변화에 대한 연구
- 장내미생물 대사를 통한 in vivo 독성평가 기술에 관한 표준작업수순서 작성

○ 연구개발에 따른 기대성과
◦ 식품 및 의약품 등의 안전성 평가를 위한 장내미생물 대사체 독성시험법의 개발 및 제공
◦ 장내미생물의 분해, 대사 작용에 의해 독성을 나타내는 식품 및 의약품 등을 평가하여 기초자료를 제공함으로써 안전관리 정책에 기여
◦ 식품 및 의약품 등의 안전성평가에 필요한 새로운 기술의 확보 및 선진화
◦ 독성학연구에 있어서 장내미생물에 의한 대사의 젓요성 인식
◦ 장내미생물 활용 독성평가 인력 및 기술 기반의 구축
◦ 장내미생물의 대사작용을 이용한 식품 및 의약품 등의 영향평가를 통해 안전관리정책에 활용가능

표/그림 (231)

표 Cell death and mitochondria
표 Intracellular signaling pathways of inflammation
표 Detection of genotoxic materials using the comet assay system.
표 Micronuclei that meet the scoring criteria
표 The composition of the human gastrointestinal microflora
표 HPLC chromatograms of arbutin and hydroquinone in BL medium.
표 HPLC chromatograms of arbutin and hydroquinone in BL medium. Bifidobacterium were cultured in the presence of arbutin for 24 h.
표 HPLC chromatograms of arbutin and hydroquinone in CM medium. B. fragilis was cultured in the presence of arbutin for 24 h.
표 HPLC chromatograms of arbutin and hydroquinone in anaerobic diluent. Bifidobacterium were cultured in the presence of arbutin for 24 h.
표 HPLC chromatograms of arbutin and hydroquinone in anaerobic diluent. Bif. longum HY81 were cultured in the presence of arbutin for 6 h and 24 h.
표 HPLC chromatograms of arbutin and hydroquinone.
표 ESI mass spectra of puerarin and daidzein acquired in the negative ion mode. (upper) A full scan mass spectra of puerarin and daidzein. (lower) MS2 spectra of puerarin and daidzein
표 LC-MS chromatogram for the determination of puerarin and daidzein in stock solution.
표 ESI mass spectra of baicalin and baicalein acquired in the negative ion mode. (upper) A full scan mass spectra of baicalin and baicalein. (lower) MS2 spectra of baicalin and baicalein
표 Concentrations of arbutin and hydroquinone in BL medium and CM medium following bacterial cultures with arbutin. Bifidobacterium and B. fragilis were cultured in the presence of arbutin for 20 h.
표 LC-MS chromatograms of baicalin and baicalein in BL medium. B. longum and Bif. adolescentis were cultured in the presence of baicalin for 20 h.
표 LC-MS chromatograms of baicalin and baicalein in CM medium. B. fragilis were cultured in the presence of baicalin for 20 h.
표 Proliferation of splenocytes in response to mitogens of different concentrations.
표 Proliferation of splenocytes in response to BL medium of different concentrations.
표 Proliferation of splenocytes in response to CM medium of different concentrations.
표 Proliferation of splenocytes in response to intestinal bacteria of different concentrations.
표 Effect of the incubation of arbutin with BL medium on the proliferation of splenocytes.
표 Effect of the incubation of intestinal bacteria with arbutin on the proliferation of splenocytes. B. longum and B. adolescentis were cultured in the presence of arbutin with BL medium for 20 h.
표 Effect of the incubation of intestinal bacteria with arbutin on the proliferation of splenocytes. Bac. fragilis was cultured in the presence of arbutin with CM medium for 20 h.
표 Effect of the incubation of sudan III with BL medium on the proliferation of splenocytes.
표 Effect of the incubation of intestinal bacteria with sudan III on the proliferation of splenocytes. B. longum and B. adolescentis were cultured in the presence of sudan III with BL medium for 20 h.
표 Effect of the incubation of intestinal bacteria with sudan III on the proliferation of splenocytes. B. fragilis was cultured in the presence of sudan III with CM medium for 20 h.
표 Effect of the incubation of baicalin with BL medium on the proliferation of splenocytes.
표 Effect of the incubation of intestinal bacteria with baicalin on the proliferation of splenocytes. B. longum and B. adolescentis were cultured in the presence of baicalin with BL medium for 20 h
표 Effect of the incubation of intestinal bacteria with baicalin on the proliferation of splenocytes. B. fragilis was cultured in the presence of baicalin with CM medium for 20 h.
표 Effect of arbutin and hydroquinone in hepatoma cells. The cells were cultured in the presence of various concentrations of arbutin and hydroquinone for 24 h. After the treatment, cell viability was measured with an MTT assay.
표 Effect of arbutin and hydroquinone in hepatoma cells. The cells were cultured in the presence of various concentrations of arbutin and hydroquinone for 24 h. Cell viability were estimated by LDH realase assay
표 Effect of geniposide and genipin in hepatoma cells. The cells were cultured in the presence of various concentrations of geniposide and genipin for 24 h. After the treatment, cell viability was measured with an MTT assay
표 Effect of geniposide and genipin in hepatoma cells. The cells were cultured in the presence of various concentrations of geniposide and genipin for 24 h. Cell viability were estimated by LDH realase assay
표 Effect of baicalin and baicalein in hepatoma cells. The cells were cultured in the presence of various concentrations of baicalin and baicalein for 24 h. After the treatment, cell viability was measured with an MTT assay and LDH realase assay.
표 Effect of puerarin and daidzein in hepatoma cells. The cells were cultured in the presence of various concentrations of puerarin and daidzein for 24 h. After the treatment, cell viability was measured with an MTT assay and LDH realase assay.
표 Effect of methylparaben and benzoic acid in hepatoma cells. The cells were cultured in the presence of various concentrations of methylparaben and benzoic acid for 24 h. After the treatment, cell viability was measured with an MTT assay and LDH realase assay.
표 Effect of arbutin and hydroquinone on the intracellular ROS formation. After the medium was removed, HepG2 cells were exposed to arbutin and hydroquinone for 2 h and fluorescence was then measured using fluorescence spectrophotometer.
표 Effect of geniposide and genipin on the intracellular ROS formation. After the medium was removed, HepG2 cells were exposed to geniposide and genipin for 2 h and fluorescence was then measured using fluorescence spectrophotometer
표 Activation of caspase-3 by arbutin and hydroquinone in HepG2 cells. Cells were pretreated with z-DEVD-fmk for 1 hr, and then exposed to arbutin and hydroquinone for additional 24 h. The catalytic activities of caspase-3 in cell lysates were assayed using the specific substrate DEVD-pNA
표 Activation of caspase-3 by geniposide and genipin in HepG2 cells. Cells were pretreated with z-DEVD-fmk for 1 hr, and then exposed to geniposide and genipin for additional 24 h. The catalytic activities of caspase-3 in cell lysates were assayed using the specific substrate DEVD-pNA
표 Effects of arbutin and hydroquinone on the expression of Bcl-2 family proteins: RT-PCR analysis of Bcl-2 family proteins in HepG2 cells. After 24 hr of incubation, the total RNA was prepared and real-time PCR was performed. GAPDH was used as a loading control
표 Effect of geniposide and genipin on the expression of Bcl-2 family proteins: RT-PCR analysis of Bcl-2 family proteins in HepG2 cells. After 24 hr of incubation, the total RNA was prepared and real-time PCR was performed. GAPDH was used as a loading control
표 Effect of arbutin and hydroquinone on apoptosis in hepatoma cells. The cells were cultured in the presence of various concentrations of arbutin and hydroquinone for 24 h. After the treatment, APOPercentage assays were performed according to the manufacturer
표 Effect of geniposide and genipin on apoptosis in hepatoma cells. The cells were cultured in the presence of various concentrations of geniposide and genipin for 24 h. After the treatment, APOPercentage assays were performed according to the manufacturer
표 Effect of arbutin in hepatoma cells. The cells were cultured in the presence of various conditions of arbutin for 24 h. After the treatment, cell viability was measured with an MTT assay
표 Effect of geniposide in hepatoma cells. The cells were cultured in the presence of various conditions of geniposide for 24 h. After the treatment, cell viability was measured with an MTT assay
표 Effect of Sudan III in hepatoma cells. The cells were cultured in the presence of various conditions of Sudan III for 24 h. After the treatment, cell viability was measured with an MTT assay
표 Effect of amaranth in hepatoma cells. The cells were cultured in the presence of various conditions of amaranth for 24 h. After the treatment, cell viability was measured with an MTT assay
표 Effect of arbutin and hydroquinone on the expression of p53. The cells were lysed and the total RNA was analyzed by RT-PCR in HepG2 cells. After 24 hr of incubation, the total RNA was prepared and PCR was performed. GAPDH was used as a loading control
표 Effect of arbutin and hydroquinone on the expression of p53 and OGG1. The cells were lysed and the total RNA was analyzed by RT-PCR in HepG2 cells. After 24 hr of incubation, the total RNA was prepared and real-time PCR was performed. GAPDH was used as a loading control
표 Effects of arbutin and hydroquinone on p53 luciferase activity in HepG2 cells. Cells were transiently co-transfected with pCMV-β-gal, p53 regulated luciferase reporter gene. After 4 h, cells were treated with the indicated concentrations of arbutin and hydroquinone for 24 h.
표 Effect of arbutin and hydroquinone on the intracellular ROS formation. After the medium was removed, HepG2 cells were exposed to arbutin and hydroquinone for 2h and fluorescence was then measured using fluorescence spectrophotometer
표 HPLC chromatogram of fecalase.
표 HPLC chromatogram of arbutin and hydroquinone.
표 HPLC chromatograms of arbutin plus fecalase.
표 Effect of arbutin plus fecalase on the expression of p53 and OGG1. The cells were lysed and the total RNA was analyzed by RT-PCR in HepG2 cells. After 24 hr of incubation, the total RNA was prepared and real-time PCR was performed. GAPDH was used as a loading control
표 Effect of geniposide and genipin on the expression of p53. The cells were lysed and the total RNA was analyzed by RT-PCR in HepG2 cells. After 24 hr of incubation, the total RNA was prepared and PCR was performed. GAPDH was used as a loading control.
표 Effect of geniposide and genipin on the expression of p53 and OGG1. The cells were lysed and the total RNA was analyzed by RT-PCR in HepG2 cells. After 24 hr of incubation, the total RNA was prepared and real-time PCR was performed. GAPDH was used as a loading control
표 Effects of geniposide and genipin on p53 luciferase activity in HepG2 cells. Cells were transiently co-transfected with pCMV-β-gal, p53 regulated luciferase reporter gene. After 4h, cells were treated with the indicated concentrations of geniposide and genipin for 24 h
표 Effect of geniposide and genipin on the intracellular ROS formation. After the medium was removed, HepG2 cells were exposed to geniposide and genipin for 2 h and fluorescence was then measured using fluorescence spectrophotometer
표 Effect of geniposide plus fecalase on the expression of p53. The cells were lysed and the total RNA was analyzed by RT-PCR in HepG2 cells. After 24 hr of incubation, the total RNA was prepared and PCR was performed. GAPDH was used as a loading control
표 Effect of geniposide plus fecalase on the expression of p53 and OGG1. The cells were lysed and the total RNA was analyzed by RT-PCR in HepG2 cells. After 24 hr of incubation, the total RNA was prepared and real-time PCR was performed. GAPDH was used as a loading control
표 Effect of baicalin and bacialein on the expression of p53 and OGG1. The cells were lysed and the total RNA was analyzed by RT-PCR in HepG2 cells. After 24 hr of incubation, the total RNA was prepared and real-time PCR was performed. GAPDH was used as a loading control.
표 Effects of baicalin and bacialein on p53 luciferase activity in HepG2 cells. Cells were transiently co-transfected with pCMV-β-gal, p53 regulated luciferase reporter gene. After 4 h, cells were treated with the indicated concentrations of baicalin and bacialein for 24 h.
표 Effect of puerarin and daidzein on the expression of p53 and OGG1. The cells were lysed and the total RNA was analyzed by RT-PCR in HepG2 cells. After 24 hr of incubation, the total RNA was prepared and real-time PCR was performed. GAPDH was used as a loading control
표 Effects of puerarin and daidzein on p53 luciferase activity in HepG2 cells. Cells were transiently co-transfected with pCMV-β-gal, p53 regulated luciferase reporter gene. After 4 h, cells were treated with the indicated concentrations of puerarin and daidzein for 24 h.
표 Effect of geniposide and genipin on the expression of COX-2. The cells were lysed and the total RNA was analyzed by RT-PCR in Raw 264.7 cells. After 24 hr of incubation, the total RNA was prepared and PCR was performed. GAPDH was used as a loading control
표 Effects of geniposide and genipin on COX-2 luciferase activity in Raw 264.7 cells. Cells were transiently co-transfected with pCMV-β-gal, COX-2 luciferase reporter gene. After 4 h, cells were treated with the indicated concentrations of geniposide and genipin for 24 h
표 Effects of geniposide and genipin on NF-kB and AP-1 luciferase activity in Raw 264.7 cells. Cells were transiently co-transfected with pCMV-β-gal, NF-kB and AP-1 luciferase reporter gene. After 4 h, cells were treated with the indicated concentrations of geniposide and genipin for 24 h.
표 Effects of geniposide plus fecalase on COX-2 luciferase activity in Raw 264.7 cells. Cells were transiently co-transfected with pCMV-β-gal, COX-2 luciferase reporter gene. After 4 h, cells were treated with the indicated concentrations of geniposide plus fecalase for 24 h. Each bar shows the mean±S.D. of three independent experiments, performed in triplicate
표 Effects of geniposide plus fecalase on NF-kB and AP-1 luciferase activity in Raw 264.7 cells. Cells were transiently co-transfected with pCMV-β-gal, NF-kB and AP-1 luciferase reporter gene. After 4 h, cells were treated with the indicated concentrations of geniposide plus fecalase for 24 h. Each bar shows the mean±S.D. of three independent experiments, performed in triplicate.
표 Effect of baicalin and bacialein on the expression of COX-2. The cells were lysed and the total RNA was analyzed by RT-PCR in Raw 264.7 cells. After 24 hr of incubation, the total RNA was prepared and PCR was performed. GAPDH was used as a loading control.
표 Effects of baicalin and bacialein on COX-2 luciferase activity in Raw 264.7 cells. Cells were transiently co-transfected with pCMV-β-gal, COX-2 luciferase reporter gene. After 4 h, cells were treated with the indicated concentrations of baicalin and bacialein for 24 h.
표 Effects of baicalin and bacialein on NF-kB and AP-1 luciferase activity in Raw 264.7 cells. Cells were transiently co-transfected with pCMV-β-gal, NF-kB and AP-1 luciferase reporter gene. After 4 h, cells were treated with the indicated concentrations of baicalin and bacialein for 24 h.
표 Effects of arbutin and hydroquinone on apoptosis in hepatoma cells. TUNEL assays were performed according to the manufacturer’s instructions. Apoptotic cells were visualized under a fluorescent microscope
표 Effect of arbutin plus fecalase on Bcl-2 and Bax expression. Total RNAs were isolated from arbutin plus fecalase treated cells and the expression of Bcl-2 and Bax were measured by real time-PCR
표 Effect of arbutin metabolism by fecalase on Bcl-2 family and caspase-3 activity in HepG2 cells. Western blotting analyses of Bax, Bcl-2 and cleaved-caspase-3 family proteins in HepG2 cells
표 Effect of arbutin metabolism on catalytic activity of caspase-3. The catalytic activities of caspase-3 in cell lysates were assayed using the specific substrate DEVD-pNA.
표 Effect of arbutin metabolism by fecalase intracellular ROS formation. Cells were seeded in 48-well plates, cultured for 24 h, and then treated with arbutin, HQ or pre-activated arbutin by intestinal microflora for 20 h. Following the incubation, cells were treated with H2DCFDA for 30 min.
표 Effect of arbutin metabolism by fecalase on Cytotoxicity. To examine the protective effect of NAC on HQ or pre-activated-induced toxicity, cells werepretreated with 1 mM NAC 1 h prior to HQ and pre-activated arbutin treatment
표 Effect of arbutin metabolism by fecalase on apoptosis in hepatoma cells. TUNEL assays were performed according to the manufacturer’s instructions. Apoptotic cells were visualized under a fluorescent microscope.
표 Effects of geniposide and genipin on apoptosis in hepatoma cells. TUNEL assays were performed according to the manufacturer’s instructions. Apoptotic cells were visualized under a fluorescent microscope
표 Effect of geniposide plus fecalase on Bcl-2 and Bax expression. Total RNAs were isolated from geniposide plus fecalase treated cells and the expression of Bcl-2 and Bax were measured by Real Time-PCR.
표 Effect of geniposide metabolism by fecalase on Bcl-2 family and caspase-3 activity in HepG2 cells. Western blotting analyses of Bax, Bcl-2 and cleaved-caspase-3 family proteins in HepG2 cells.
표 Effect of geniposide metabolism on catalytic activity of caspase-3. The catalytic activities of caspase-3 in cell lysates were assayed using the specific substrate DEVD-pNA.
표 Effect of geniposide metabolism by fecalase intracellular ROS formation. Cells were seeded in 48-well plates, cultured for 24 h, and then treated with geniposide, genipin or pre-activated geniposide by intestinal microflora for 20 h. Following the incubation, cells were treated with H2DCFDA for 30 min.
표 Effect of geniposide metabolism by fecalase on Cytotoxicity. To examine the protective effect of NAC on genipin or pre-activated-induced toxicity, cells werepretreated with 1 mM NAC 1 h prior to genipin and pre-activated geniposide treatment
표 Effect of geniposide metabolism by fecalase on apoptosis in hepatoma cells. TUNEL assays were performed according to the manufacturer’s instructions. Apoptotic cells were visualized under a fluorescent microscope.
표 Effects of activated geniposide on the JNK pathway. Western blotting analyses of p-JNK, p-ERK, and p-p38 proteins.
표 Effect of geniposide metabolism by fecalase on Cytotoxicity. To examine the protective effect of SP600125 on genipin or pre-activated-induced toxicity, cells were pretreated with 1 mM SP600125 1 h prior to genipin and pre-activated geniposide treatment.
표 Effects of activated geniposide on the JNK pathway. TUNEL assays were performed according to the manufacturer’s instructions. Apoptotic cells were visualized under a fluorescent microscope.
표 Effect of cytotoxic activity of arbutin by intestinal microflora. The cells were cultured in the presence of various concentrations of arbutin for 24 h. After the treatment, cell viability was measured with an MTT assay.
표 Effect of cytotoxic activity of arbutin by intestinal microflora. The cells were cultured in the presence of various concentrations of arbutin for 24 h. After the treatment, cell viability was measured with an LDH realase assay.
표 Effects of arbutin and its metabolites on apoptosis in HepG2 cells. The cells were cultured in the presence of various concentrations of arbutin and its metabolites for 24 h. After the treatment, APOPercentage assays were performed according to the manufacturer
표 Effect of arbutin plus fecalase and microbial enzyme mix on Bcl-2 and Bax expression. Total RNAs were isolated from arbutin and its metabolites treated cells and the expression of Bcl-2 and Bax were measured by real time-PCR.
표 Effect of arbutin metabolism on catalytic activity of caspase-3. The catalytic activities of caspase-3 in cell lysates were assayed using the specific substrate DEVD-pNA
표 Effect of arbutin metabolism by fecalase and microbial enzyme mix intracellular ROS formation. Cells were seeded in 48-well plates, cultured for 24 h, and then treated with arbutin and its metabolite for 20 h. Following the incubation, cells were treated with H2DCFDA for 30 min.
표 Effect of cytotoxic activity of geniposide by intestinal microflora. The cells were cultured in the presence of various concentrations of geniposide and its metabolites for 24 h. After the treatment, cell viability was measured with an MTT assay.
표 Effect of cytotoxic activity of geniposide by intestinal microflora. The cells were cultured in the presence of various concentrations of geniposide and its metabolites for 24 h. After the treatment, cell viability was measured with an LDH realase assay.
표 Effect of geniposide and its metabolites on apoptosis in HepG2 cells. The cells were cultured in the presence of various concentrations of geniposide and its metabolites for 24 h. After the treatment, APOPercentage assays were performed according to the manufacturer
표 Effect of geniposide metabolism by fecalase and microbial enzyme mix on Bcl-2 family and caspase-3 activity in HepG2 cells. Western blotting analyses of Bax, Bcl-2 and cleaved-caspase-3 family proteins in HepG2 cells.
표 Effect of geniposide plus fecalase and microbial enzyme mix on Bcl-2 and Bax expression. Total RNAs were isolated from geniposide and its metabolites treated cells and the expression of Bcl-2 and Bax were measured by real time-PCR.
표 Effect of geniposide metabolism on catalytic activity of caspase-3. The catalytic activities of caspase-3 in cell lysates were assayed using the specific substrate DEVD-pNA
표 Effect of geniposide metabolism by fecalase and microbial enzyme mix intracellular ROS formation. Cells were seeded in 48-well plates, cultured for 24 h, and then treated with geniposide and its metabolite for 20 h. Following the incubation, cells were treated with H2DCFDA for 30 min.
표 Effect of cytotoxic activity of bacalin by intestinal microflora. The cells were cultured in the presence of various concentrations of bacalin and its metabolites for 24 h. After the treatment, cell viability was measured with an MTT assay.
표 Effect of cytotoxic activity of methyl-paraben by intestinal microflora. The cells were cultured in the presence of various concentrations of methyl-paraben and its metabolites for 24 h. After the treatment, cell viability was measured with an MTT assay.
표 Effect of arbutin and its metabolites on DNA demage in HepG2 cells. DNA damage was measured by comet assay in HepG2 cells treated with arbutin and its metabolites for 24 h.
표 Micronuclei in binucleate HepG2 cells. Cells were seeded in 6 well plate. Following 24-h treatment the cells were blocked at cytokinesis by the addition of fresh medium containing 3 mg/ml cytochalasin B. Cells were cultured for a further 24 h prior to fixation and staining with 5% Giemsa.
표 Comparison of the micronucleated cells in HepG2 cell in control and various treatment groups. Following 24-h treatment the cells were blocked at cytokinesis by the addition of fresh medium containing 3 mg/ml cytochalasin B. Micronucleus formation was scored in 1000 binucleate cells either as micronucleated binucleate cells or total number of micronuclei
표 Effect of geniposide and genipin on the expression of p53. Western blotting analyses of p53 and beta-actin proteins in HepG2 cells.
표 Effects of geniposide plus fecalase and and microbial enzyme mix on p53 luciferase activity. Cells were transiently co-transfected with pCMV-β-gal, p53 luciferase reporter gene. After 4 h, cells were treated with the indicated concentrations of geniposide and its metabolite for 24 h.
표 Effect of geniposide and its metabolites on DNA demage in HepG2 cells. DNA damage, measured by comet assay in HepG2 cells treated with geniposide and its metabolites for 24 h.
표 Micronuclei in binucleate HepG2 cells. Cells were seeded in 6 well plate. Following 24-h treatment the cells were blocked at cytokinesis by the addition of fresh medium containing 3 mg/ml cytochalasin B. Cells were cultured for a further 24 h prior to fixation and staining with 5% Giemsa.
표 Comparison of the micronucleated cells in HepG2 cell in control and various treatment groups. Following 24-h treatment the cells were blocked at cytokinesis by the addition of fresh medium containing 3 mg/ml cytochalasin B. Micronucleus formation was scored in 1000 binucleate cells either as micronucleated binucleate cells or total number of micronuclei
표 Effects of geniposide plus fecalase and and microbial enzyme mix on COX-2 luciferase activity in Raw 264.7 cells. Cells were transiently co-transfected with pCMV-β-gal, COX-2 luciferase reporter gene. After 4 h, cells were treated with the indicated concentrations of geniposide and its metabolite for 24 h.
표 Effects of geniposide plus fecalase and and microbial enzyme mix on AP-1 and NF-kB luciferase activity in Raw 264.7 cells. Cells were transiently co-transfected with pCMV-β -gal, AP-1 and NF-kB luciferase reporter gene. After 4 h, cells were treated with the indicated concentrations of geniposide and its metabolite for 24 h.
표 분변의 희석배수효과
표 초음파처리 시간에 따른 효소활성의 비교
표 초음파 처리하여 원심분리효과
표 우리나라 사람의분변의 효소활성
표 우리나라 사람의분변의 효소활성
표 우리나라 사람의분변의 효소활성
표 우리나라 사람의 장내세균총에 의한 천연성분의 대사활성
표 우리나라 사람의 장내세균총에 의한 약물 대사활성
표 우리나라 사람의 방부제 및 염색시약의 장내세균총 의한 대사활성
표 Fecal enzyme activities of Koreans
표 The difference of fecal bacterial enzyme activities between males and females.
$Average metabolic activity of acetaminophen and digoxin by human fecal enzyme mix fraction.$ 표 Average metabolic activity of acetaminophen and digoxin by human fecal enzyme mix fraction.
표 The difference of fecal bacterial enzyme activities metabolizing acetaminophen and digoxin between individuals
표 The difference of fecal bacterial enzyme activities metabolizing acetaminophen and digoxin between males and females
$Average metabolic activity of herbal medicine constituents by human fecal enzyme mix fraction.$ 표 Average metabolic activity of herbal medicine constituents by human fecal enzyme mix fraction.
$Metabolic activity of herbal medicine constituents by human fecal enzyme mix fraction.$ 표 Metabolic activity of herbal medicine constituents by human fecal enzyme mix fraction.
표 The difference of fecal bacterial enzyme activities metabolizing herbal medicine constituents between males and females
표 Rutin 및 quercetin의 돌연변이원성에 분변의 fecalase 및 S9 mix 처리 효과
표 Metabolic activity of ginsenoside Rb1 isolated from ginseng (left) and in ginseng extract (right) by Korean fecal enzyme mix.
표 Metabolic activity of baicalin isolated from Scutellariae Radix (black circle) and in Scutellariae radix extract (yellow triangle) by Korean fecal enzyme mix. Data of left and right columns are the metabolic activities of male and female in each age.
표 Metabolism of baicalin by human fecal microflora. Identification of baicalin isolated (a) and baicalin metabolites with LC-MS/MS; (i) baicalin, (ii) baicalein, and (iii) oroxylin A.
표 Metabolic activity of baicalin (closed circle) by Korean fecal enzyme mix. The metabolites, produced from baicalin were baicalein (open square) and oroxylin A (closed triangle). Data of left and right columns are the metabolic activities of male and female in each age.
표 Inhibitory effects of orally administered baicalin, its metabolites, and azelastine, on scratching behavior induced by histamine or compound 48/80 in mice. The scratching behavior was induced by histamine and compound 48/80 in ICR and BALB/c mice, respectively. (up) Effect of baicalin, baicalein, oroxylin A and azelastine in histamine-induced scratching behavior in ICR mice. (down) Effect of baicalin, baicalein, oroxylin A and azelastine on compound 48/80-induced scratching behavior in BALB/c mice. These agents [10 (gray bar), 20 (white bar) and 50 mg/kg (black bar)] were administered orally 1 or 5 h before the intradermal injection of histamine or compound 48/80. The frequency of scratching behavior in the normal group treated with saline alone for 1 h were 2 ± 1, respectively, and the frequency of scratching behavior in the control group treated with histamine in ICR mice and compound 48/80 in BALB/c mice was 89 ± 5 and 168 ± 18, respectively
표 Inhibitory effects of baicalin, its metabolites, and azelastine administered intraperitonelly on histamine-induced scratching behavior in mice. The scratching behavior was induced by histamine in ICR mice. Mice were treated with or without intraperioneal administration of the test agents [5(gray bar), 10 (white bar) and 20 mg/kg (black bar)] 1 or 5 h before the intradermal injection of 300 μg/ml of histamine into the skin on the backs of mice. The frequency of scratching behavior in the normal (treated with saline alone) and control groups (treated with histamine) for 1 h was 2 ± 1 and 85 ± 4, respectively.
표 Inhibitory effects of baicalin, baicalein, and azelastine administered orally on histamine-induced scratching behavior in mice treated with or without antibiotics. The scratching behavior was induced by histamine in ICR mice treated with or without antibiotics. The antibiotics were administered orally 1 day before histamine treatment. Oral administration of test agents [10(gray bar), 20 (white bar), and/or 50 mg/kg (black bar)] was treated 1 h before the intradermal injection of 300 μg/50 μl of histamine into the skin on the backs of mice. The frequency of scratching behavior in the normal group treated with saline alone in normal and antibiotics-treated mice for 1 h were 2 ± 2 and 2 ± 1, respectively, and the frequency in the control group treated with histamine in normal and antibiotics-treated mice was 84 ± 4 and 87 ± 5, respectively. Ba, baicalin; ABa, baicalin with antibiotics; Be, baicalein; ABe, baicalein with antibiotics; Oa, oroxylin; AOa, oroxylin A with antibiotics; A, azelasitine; AA azelastine with antibiotics
표 Inhibitory effects of baicalin, its metabolites, and azelastine administered orally on vascular permeability increased by histamine in mice. (A) Effect in vascular permeability. The vascular permeability was increased by histamine in ICR mice. Mice were treated with or without the oral administration of test agents [10 (gray bar), 20 (white bar), and/or 50 mg/kg (black bar)] 1 h before the intradermal injection of 300 mg/50 ml of histamine into the skin on the backs of mice. In the vascular permeability assay, the amount of Evan blue extravasated from the dorsal skin (1 x 1 cm) of the control group stimulated with histamine and the vehicle-treated group was 12 ± 3 μg and 4 ± 2 μg, respectively. The values indicate the mean ± S.D. (n=6). * P< 0.05 versus baicalin (50mg/kg) treated group. (B) Histologic photograph. The skin tissues were stained with hematoxylin–eosin and assessed by light microscopy
표 Anti-histamine effect of baicalin, its metabolites, and azelastine in Magnus test using guinea pig ileum. The ileal strip was set in a 10 ml Magnus tube (32oC,95%O2+5%CO2) containing Tyrode solution. Each test agent [0.1 mM (gray bar) and 0.4 mM (blackbar), dissolved in 2% trition X-100] was added to the preparation 30 s before treatment with histamine (1x10-6M).The values indicate mean ± S.D. (n=3). #P < 0.05 versus 0.1 mM baicalin treated group, * P< 0.05 versus 0.4 mM baicalin treated group.
표 Mean plasma concentration–time profile of lancemaside A and its metabolites in mice after oral administration of lancemaside A (60 mg/kg). Lancemaside A, lancemaside X and EAG were not detected, but echinocystic acid (closed square) was detected. (each point represents mean±S.D., n = 4). Structures of lancemaside A (A) and its metabolites, lancemaside X (B), echinocystic acid 3-O-β -D-glucuronopyranoside (EAG, C) and echinocystic acid (D).
표 ESI-MA analysis of lancemaside A and its metabolites in the digestive tract contents of mice orally administered lancemaside A. Three main peaks were detected: The ion peak at m/z 1189 is lancemasideA, another ion peak at m/z 1014 is lancemaside X, the other ion peak at m/z 471 is echinocysticacid
표 Metabolism of lancemaside A by human fecal suspension.
표 Fecal enzyme activities of Koreans
$Average metabolic activity of acetaminophen, bisacodyl, digoxin, methylparaben and natural product constituents by human fecal enzyme mix fraction$ 표 Average metabolic activity of acetaminophen, bisacodyl, digoxin, methylparaben and natural product constituents by human fecal enzyme mix fraction
표 Effect of fat diet on fecal bacterial enzyme activities in mice.
표 Fecal compound K-forming activities from ginsenoside Rb1 and ginseng extract of 100 Koreans.
표 Metabolic activities of ginsenoside Rb1 and Rg1 by the feces of 100 Koreans.
표 Profile of the relationship between fecal enzyme activities.
표 HPLC analysis of biotransform of Ginsenoside Rb1. We detected HPLC patterns at 1h, 3h and 6h after oral administered ginsenoside Rb1 (Rb1, 60 mg/kg) with or without antibiotics (streptomycin and neomycin, 100 mg/kg) in cecum and colon. Ginsenoside Rb1 was biotransformed to compound K (CK) in mice without administered antibiotics. However, in mice with administered antibiotics, compound K was not detected
표 Inhibitory effects of ginsenosides in TNBS-induced colitic mice. Ginsenoside Rb1 (Rb1) and compound K (CK) (10 mg/kg or 20 mg/kg), sulfasalazine (SUL, 50 mg/kg), or saline was orally administered for 3 days after TNBS treatment. Mice were sacrificed 12 h after the final administration of test agents
표 Effects of ginsenoside Rb1 and compound K on inflammatory cytokines in TNBS-induced colitic mice.
표 Inhibitory effects of ginsenoside Rb1 and compound K on the productions of pro-inflammatory cytokines in LPS-induced peritoneal macrophages. All data are expressed as means ± SDs (n = 4 in a single experiment). #p<0.05, significantly different vs. normal control group. *p<0.05, vs. LPS control.
표 Effects of ginsenoside Rb1 and compound K on phosphorylation of IKK-β, activations of NF- κB and IRAK-1, and interaction between LPS and TLR-4.
표 Inhibitory effects of ginsenoside Rb1 or compound K on IRAK-1 kinase binding activity.
표 Baicalin-metabolic activities between individuals (closed symbols, male; open symbols, females).
표 Effect of baicalin and its metabolites baicalein and oroxylin A on LPS-stimulated systemic inflammation in mice.
표 Effect of baicalin and its metabolites baicalein and oroxylin A on LPS-stimulated systemic inflammation in mice.
표 Effect of orally administered antibiotics on the enzyme activity of intestinal microflora in mice.
표 The enzyme productivity of intestinal bacteria isolated from Koreans.
표 Comparison between Korean average fecalase and intestinal bacterial enzyme mix activities.
표 Stability of intestinal bacterial emzyme mix.
표 Effect of Sophorae flos extract and its main constituent rutin on S. typimurium TA98 reverse mutation assay. NC, negative control; M, test agent alone; M+EM, agent + fecal microbial enzyme mix; M+F, agent + fecalase; M+S9, agent + S9 mix; M+EM+S9, agent + fecal microbial enzyme mix + S9 mix; M+F+S9, agent + fecalase + S9 mix. The mutant frequency is expressed as the number of revertant colonies. The mutant potential of the test is assumed if the mutant frequency is 2-fold higher than negative control
표 Effect of Sophorae flos extract and its main constituent rutin on S. typimurium TA100 reverse mutation assay.
표 Effect of Sophorae flos extract and its main constituent rutin on S. typimurium TA102 reverse mutation assay.
표 Effect of Sophorae flos extract and its main constituent rutin on S. typimurium TA1535 reverse mutation assay.
표 Effect of Sophorae flos extract and its main constituent rutin on S. typimurium TA1537 reverse mutation assay.
표 Hard isolator
표 HPLC gradient program
표 Mobile phase gradient for analysis of baicalin and its metabolite in biological samples
표 Condition of LC/ESI-MS for analysis of baicalin and its metabolite in biological samples
표 Mobile phase gradient for analysis of baicalin and its metabolite in biological samples
표 Gnotobiote production by oral feeding of human fecal suspension.
표 Blood biochemistry results after oral administration of ginsenoside Re in germ-free and SPF mice
표 Histologic diagnosis after oral administration of ginsenoside Re in germ-free and SPF mice.
표 Chromatgrams of ginsenoside Re and its relevant metabolites
표 Plasma concentration (ng/ml) after oral adminstration of ginsenoside Re to germ-free and SPF mice
표 Plasam concentration of ginsenoside Re after oral administration to germ-free and SPF mice
표 Pharmacokientic paramters for ginsenoside Re in the germ-free and SPF mice
표 Blood biochemistry results after oral administration of acetaminophen in germ-free, gnotobiote and SPF mice.
표 Histologic diagnosis after oral administration of acetaminophen in germ-free, gnotobiote and SPF mice.
표 Histopathological findings in SD rats
표 Histologic diagnosis after oral administration of baicalin in normal and antibiotic-treated rats. ×100, H&E stain. There was no remarkable change.
표 Microbial enumeration using anaerobic culture of intestinal contents after oral administration of antibiotics for 3 days.
표 Representative full mass chromatograms and MS spectra of baicalin and its metabolites in rat serum after oral administration with 100 mg/kg baicalin.
표 MS/MS chromatograms and MS/MS spectra of baicalin and its metabolites in rat serum after oral administration with 100 mg/kg baicalin.
표 SRM chromatograms obtained with serum sample after oral administration of baicalin at 100 mg/kg.
표 The proposed metabolic pathway of baicalin in rat serum after oral administration of baicalin.
표 Accuracy and precision of determination for baicalin in rat serum
표 Mean serum concentration-time curves after oral administration of baicalin (100mg/kg) in normal and antibiotic-treated rats.
표 Pharmacokinetic parameters of baicalin after oral administration of baicalin (100 mg/kg) in normal and antibiotic-treated rats.
표 Mean serum concentration-time curves of baicalin and its major metabolites after oral administration of baicalin (100 mg/kg) in normal and antibiotic-treated rats.
표 Mean concentration of baicalin and its major metabolites obtained with in vitro metabolism of baicalin (50 μM) by intestinal content microflora.
표 Mean concentration of metabolites obtained with in vitro metabolism of baicalin (50 μM) by intestinal content microflora.
표 MS/MS chromatograms and spectra of metabolite M4 and M5 obtained with in vitro metabolism of baicalin (50 μM) by intestinal content microflora.
표 MS/MS chromatograms of metabolite M4 obtained with in vitro metabolism of baicalin (50 μM) by intestinal content microflora.
표 MS/MS chromatograms of metabolite M5 obtained with in vitro metabolism of baicalin (50 μM) by intestinal content microflora
표 MS/MS chromatograms and spectra of metabolite M6 and M7 obtained with in vitro metabolism of baicalin (50 μM) by intestinal content microflora
표 MS/MS chromatograms of metabolite M6 obtained with in vitro metabolism of baicalin (50 μM) by intestinal content microflora.
표 MS/MS chromatograms of metabolite M7 obtained with in vitro metabolism of baicalin (50 μM) by intestinal content microflora.
표 The proposed metabolic pathway of baicalin obtained with in vitro metabolism of baicalin (50 μM) by intestinal content microflora.
표 Gnotobiote production by oral feeding of human fecal suspension.
표 Blood biochemistry results after oral administration of acetaminophen in germ-free, gnotobiote and SPF mice (1차 동물실험 결과)
표 Blood biochemistry results after oral administration of acetaminophen in germ-free, gnotobiote and SPF mice (2차 동물실험 결과)
표 (Liver) Histologic diagnosis after oral administration of acetaminophen in germ-free, gnotobiote and SPF mice. Central lobular necrosis (arrow head) were detected in high dose of acetaminophen treated animal groups. Other dosage and other tissues didn
표 (Kidney) Histologic diagnosis after oral administration of acetaminophen in germ-free, gnotobiote and SPF mice. Each tissue doesn
표 (Lung) Histologic diagnosis after oral administration of acetaminophen in germ-free, gnotobiote and SPF mice. Each tissue doesn
표 Blood biochemistry results after oral administration of geniposide in germ-free, gnotobiote and SPF mice (1차 동물실험 결과)
표 Blood biochemistry results after oral administration of geniposide in germ-free, gnotobiote and SPF mice (2차 동물실험 결과)
표 (Liver) Histologic diagnosis after oral administration of geniposide in germ-free, gnotobiote and SPF mice. Each tissue doesn
표 (Kidney) Histologic diagnosis after oral administration of geniposide in germ-free, gnotobiote and SPF mice. Each tissue doesn
표 (Lung) Histologic diagnosis after oral administration of geniposide in germ-free, gnotobiote and SPF mice. Each tissue doesn
표 Blood biochemistry results after oral administration of baicalin in germ-free, gnotobiote and SPF mice (1차 동물실험 결과)
표 Blood biochemistry results after oral administration of baicalin in germ-free, gnotobiote and SPF mice (2차 동물실험 결과)
표 (Liver) Histologic diagnosis after oral administration of baicalin in germ-free, gnotobiote and SPF mice. Each tissue doesn
표 (Kidney) Histologic diagnosis after oral administration of baicalin in germ-free, gnotobiote and SPF mice. Each tissue doesn't show any pathological changes.
표 (Lung) Histologic diagnosis after oral administration of baicalin in germ-free, gnotobiote and SPF mice. Each tissue doesn't show any pathological changes.
표 Mean serum concentration-time curves after oral administration of baicalin (100 mg/kg) in germ-free, gnotobiote and SPF mice (1차 동물실험 결과).
표 Mean serum concentration-time curves of baicalin and its major metabolites after oral administration of baicalin (100 mg/kg) in germ-free, gnotobiote and SPF mice (1차 동물실험 결과).
표 Mean serum concentration-time curves after oral administration of baicalin (100 mg/kg) in germ-free, gnotobiote and SPF mice (2차 동물실험 결과).
표 Mean serum concentration-time curves of baicalin and its major metabolites after oral administration of baicalin (100 mg/kg) in germ-free, gnotobiote and SPF mice (2차 동물실험 결과).
표 Histopathological findings in male ICR mice (Group summary)
표 Histologic diagnosis after oral administration of baicalin in normal and antibiotic-treated mice. ×100, H&E stain. There was no remarkable change.
표 Effects of antibiotic treatment on the number of intestinal bacteria in male ICR mice. Microbial enumeration using anaerobic culture of intestinal contents were performed after oral administration of antibiotics for 3 days.
표 MRM chromatograms obtained with serum sample after administration of baicalin at 100 mg/kg.
표 Mean serum concentration-time curves after oral administration of baicalin (100 mg/kg) in normal and antibiotic-treated mice.
표 Mean serum concentration-time curves of baicalin and its major metabolites after oral administration of baicalin (100 mg/kg) in normal and antibiotic-treated mice.

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