발간등록번호 11-1543000-002003-01 상황버섯으로유도된 FMO3 와 Corin 에의한 농약분해및혈압저하유도
농림축산식품부장관귀하 본보고서를 상황버섯으로유도된 FMO3 와 Corin 에의한농약분해및혈압저하유도 ( 개발기 간 :2014. 09. ~ 2017. 09.) 과제의최종보고서로제출합니다. 2017. 11.. 주관연구기관명 : 서울시립대학교산학협력단 ( 인 ) 주관연구책임자 : 김하원 국가연구개발사업의관리등에관한규정제 18 조에따라보고서열람에동의 합니다. - 1 -
보고서요약서 과제고유번호 114074-3 해당단계 연구기간 단계구분 3 년 /3 년 연구사업명 단위사업 사업명 농식품기술개발사업 연구과제명 대과제명 세부과제명 해당단계 참여 연구원수 총 : 6 명 내부 : 5 명 외부 : 1 명 해당단계 연구개발비 정부 : 50,000천원민간 : 0천원계 : 50,000천원 연구책임자 김하원 총연구기간 총 : 8 명 정부 : 150,000 천원 참여 내부 : 7 명 총연구개발비 민간 : 0 천원 연구원수 외부 : 1 명 계 : 150,000 천원 연구기관명및 소속부서명 서울시립대학교생명과학과 참여기업명 : ( 해당없음 ) 위탁연구연구기관명 : ( 해당없음 ) 연구책임자 : ( 해당없음 ) 상황버섯의베타-글루칸을마우스에경구투여하였을때간조직에서 FMO, Corin, Metallothionein의 3종의유전자가투여일수에비례하여증가하였다. 5종의 FMO 유전자중에서 FMO2, FMO3, FMO4 가크게증가하였으며대표적으로 FMO3가증가되어동물에서소변중의악취물질인 TMA 분해촉진과생체외래물질 (xenobiotics) 인 carbendazim 농약분해촉진을증명하였다. 베타-글루칸에의하여증가된 Corin은 ANP를생산시켜새로운항암작용기전을밝혔으며, 신장에서나트륨의배설을촉진시킴을입증하여혈압저하기전을증명하였다. 증가된 Metallothionein 에의하여체내의중금속인 Pb와 Cd의배설촉진을증명하였다. 보고서면수 : 104 면 - 2 -
연구의 목적및내용 연구개발성과 연구개발성과의활용계획 ( 기대효과 ) 코드번호 D-01 국내농가의소득을향상시키고국내에서주로재배하는상황버섯 (Phellinus baumii) 의부가가치를높이기위하여, 상황버섯의베타-글루칸에대하여새로운접근방법으로연구하였다. 지금까지버섯의베타-글루칸의주요작용은면역촉진과항암작용이었다. 그러나본연구자는상황버섯의베타-글루칸의새로운효능을발견하기위하여베타-글루칸을마우스에경구투여하여크게증가하는유전자를발견하여그효능을증명하고자하였다. 상황버섯의베타-글루칸을마우스에경구투여한후에간조직으로부터총 mrna를분리하여마이크로어레이법으로분석하여크게증가된유전자 3종을발견하였다. 증가된유전자를리얼타임 PCR로재확인하였을때도동일하게증가함을증명하였다. 3종의유전자에대하여그유전자의기능을실험동물을통하여증명함으로로체계적인연구가완성되었다. 상황버섯추출물또는베타-글루칸분획에대하여새로운효능을발견하기위하여마우스에경구투여한후에간조직을추출하여마이크로어레이법으로크게증가된유전자는 FMO, Corin, Metallothionein의 3종유전자임을밝혔다. 베타-글루칸을경구투여후리얼타임 PCR분석에의하여 FMO 유전자중에서 FMO2, FMO3, FMO4 가증가하였으며대표적으로 FMO3가크게증가됨을밝혔다. 증가된 FMO 유전자는마우스소변중의악취물질인 TMA의농도가감소하였을뿐만아니라체내로주입한농약인 carbendazim의혈청내농도도신속하게감소시켰다. 베타-글루칸에의하여증가된 Corin 효소는 pro-ano를 ANP로전환시킴을밝혀냈다. 증가된 ANP는 Wnt 수용체에결합하여항암작용을나타내는기전이됨을밝혔다. 지금까지는베타-글루칸이체내의면역을증강시켜항암작용을나타낸다고알려져왔으나, 또다른항암기전으로 Corin효소를증가시켜항암작용을나타냄을밝혔다. 증가된 Corin효소의또다른작용으로신장에서나트륨의배설을촉진시킴을입증하여혈압저하기전을증명하였다. 베타-글루칸을경구투여한후에간에서 Metallothionein (MT) 단백질이증가함을리얼타임 PCR로증명하였으며, 증가된 MT에의하여마우스의각종조직에서 Pb와 Cd의배설을촉진시킴을증명하였다. 본과제를통하여세계최초로베타-글루칸이 FMO, corin, metallothionein을증가시킴을발견하는성과를이룩하게되었다. 본과제를통하여상황버섯의새로운작용을밝혔으므로아래와같은다양한실용화가달성될수있다. 1) FMO3 관련 : 인체농약제거제, 니코틴분해촉진제, 오리농장의악취감소제, 애완견의냄새제거제등의제품개발. 2) Corin 관련 : 무독성혈압저하제개발, 심장마비예방제개발, 항암제개발. 3) MT 관련 : 납또는카드뮴등의생체내유해중금속제거제개발. 그러므로, 상황버섯의베타-글루칸에대하여새로운효능을발견하였으므로국내버섯재배농가의소득증대에크게기여할것으로예상된다. 중심어 (5 개이내 ) Corin - 3 -
코드번호 D-02 Purpose& Contents In order to improve income of domestic farm house and to enhance value of cultivated mushroom products of Phellinus baumii, we investigated new approach by using beta-glucan from the mushroom. Most of the reported functions of beta-gluca has been immunostimulation and anticancer activity. However, to find novel functions of beta-glucan from Phellinus baumii, we approached finding increased genes and prove the efficacy of the induced genes by the oral administered beta-glucan in mouse. Total mrnas were isolated from mouse liver tissue and found three increased genes by the microarray method. The augmented genes were also reconfirmed by realtime PCR analysis. The new functions by the increased genes were completed systemically in experimental animal. Results We found three genes of FMO, corin and metallothionein from the mouse liver tissue that was orally administered beta-glucan. Among the FMO family, realtime PCR analysis showed that FMO2, FMO3 and FMO4 isotypes were increased, especially FMO3 was most strongly augmented. The induced FMOs decreased odor compound of TMA in urine and increased excretion of pesticide from the serum. We found increase of ANP by the induced corin enzyme. The binding of ANP to Wnt receptor could be novel antitumor mechanism. The increased corin enzyme augmented secretion of Na + from the kidney, showing an antihypertensive activity. Orally administered beta-glucan also promoted excretion of heavy metals of Pb and Cd metals in mouse organs. We are the first reporter in the world induction of FMO, corin, metallothionein genes by beta-glucan in this project. As we found novel functions of Phellinus baumii in this project, the following applications could be achieved: Expected Contribution 1) FMO3 field: Rapid clearance of pesticides, rapid nicotine clearing agent, deodorizer in pet and livestock raiser. 2) Corin field: Development of non-toxic antihypertensive drug, heart attack preventing drug and antitumor agent. 3) MT field: Development of toxic heavy metal scavenger including lead or cadmium. Therefore, it is expected to contribute greatly to increase income of domestic mushroom grower due to the finding new functions of beta-glucan from Phellinus baumii. Keywords Corin - 4 -
Chapter 1: Overview of Research and Development Project... 7 Chapter 2: Research Trends of Technic Development in Domestic... 9 Chapter 3: Methods and Results of the Accomplished Research and Development.. 20 Chapter 4: Degree of Object Achievements and Degree of Contribution to Related Field... 75 Chapter 5: Achievement of Research and Development and Plan for Achievement Application... 78 Chapter 6: Obtained Foreign Informations on Scientific Techniques during Research and Development... 80 Chapter 7: Security Level of the Achievement of Research and Development... 81 Chapter 8: Present List of Research Facilities and Equipments... 82 Chapter 9: Safeguard Performance in Laboratory during Research and Development Project... 83 Chapter 10: Typical Research Achievement List on the Research and Development Project... 84 Chapter 11: Miscellaneous... 85 Chapter 12: References... 86-5 -
제 1 장연구개발과제의개요... 7 제 2 장국내의기술개발현황... 9 제 3 장연구개발수행내용및결과... 20 제 4 장목표달성도및관련분야에의기여도... 75 제 5 장연구개발성과및성과활용계획... 78 제 6 장연구개발과정에서수집한해외과학기술정보... 80 제 7 장연구개발성과의보안등급... 81 제 8 장연구시설 장비현황... 82 제 9 장연구개발과제수행에따른연구실등의안전조치이행실적... 83 제 10 장연구개발과제의대표적연구실적... 84 제 11 장기타사항... 85 제 12 장참고문헌... 86-6 -
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제 3 장연구개발수행내용및결과 코드번호 D-05 Scheme 1. Annual plan of the project on the functions of FMOs, corin and metalothionein which were induced by the oral injection of beta-glucan from Phellinus baumii in mice. - 20 -
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Gene (Tissue used) FMO1 (Kidney) FMO1 (Kidney) FMO2 (Lung) FMO2 (Lung) FMO3 (Liver) FMO3 (Liver) FMO4 (Kidney) FMO4 (Kidney) FMO5 (Liver) FMO5 (Liver) PCR product (bp) Direction Primer sequence (5' 3') 217 F CAT TCC AAC TAC AAG GAC TCG 217 R TGT CTC TGG ACA GTG GGA AGT 382 F CCG GGT CTT TAA GGG TTT CAG G 382 R AGG CTC CAT CTT CCC AAC CGT A 102 F CAG CAT TTA CCA ATC GGT CTT C 102 R TTG CTG TGA TGC ATG AAG TTG 144 F TCC TGA GCC CAC ATT TAC CTC 144 R CCA GTG TTT CCA AGA CCA ACC 235 F GCT TGC CTA CAC GGT TCA AG 235 R ATC ACA CGG ATG CTC ACC TG GAPDH 302 F AGC CTC GTC CCG TAG ACA AA GAPDH 302 R CAC GAC ATA CTC AGC ACC GGC - 23 -
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Oral Administration Control vs 100 mg/kg PBE Cutoff (Fold change) 1.3 1.5 2 3 Liver Gene Expression No. of Modulated Gene* SOA MOA UP 1184 1358 DOWN 1264 1153 UP 328 368 DOWN 292 304 UP 45 87 DOWN 61 51 UP 8 23 DOWN 3 11-26 -
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No Gene Symbol Biological Function Average Fold Change* 1 FMO3 Drug metabolic process 5.1 17 2 MT2 Cellular zinc ion homeostasis Detoxification of copper ion SOA MO 0.9 11 3 CYP46A1 Llipid, steroid and cholesterol metabolic process 1.9 8. 4 CORIN 5 MT1 Peptide hormone processing Neuron differentiation Cellular metal and zinc ion homeostasis Nitric oxide mediated signal transduction Detoxification of copper ion 0.4 5. 1.0 4. 6 AKR1E1 Oxidation-reduction process 0.7 4. 7 PLK3 Protein phosphorylation, signal transduction 0.6 3. 8 FMO2 Organic acid metabolic process NADP metabolic process Xenobiotic, toxin and drug metabolic process 1.5 3. 9 DPY19L3 Biological process 0.6 3. 10 CML3 Negative regulation of cell adhesion 2.1 3. 11 CYP2A4 Steroid and drug catabolic process Oxidation-reduction process 1.6 3. 12 CML3 Negative regulation of cell adhesion 2.1 3. 13 GM14403 Biological process 1.5 3. 14 NIPAL1 Ion transport, magnesium ion transport 1.1 3. 15 PER1 Regulation of transcription 1.1 3. Circadian rhythm 16 CYP2A5 Oxidation-reduction process 1.5 3. 17 GM4956 Unknown 1.1 3. 18 CML3 Negative regulation of cell adhesion 2.1 3. 19 SLC16A5 Biological process 1.4 3. 20 FMO4 Oxidation-reduction process 1.3 3. 21 CXCL10 Defense response to virus Inflammatory response, immune response 1.1 3. 22 CUX2 DNA-dependent, regulation of transcription 1.4 3. 23 1810046K0 Biological process 0.6 7RIK 3. - 28 -
No Gene Symbol 1 GDPD3 2 ILDR2 Biological process Glycerol metabolic process, lipid metabolic process Response to glucose stimulus Insulin secretion Pancreas development Homeostasis of number of cells within a tissue Average Fold Change* SOA MOA 1.1 0.1 1.0 0.1 3 CFHR1 Biological process 0.8 0.2 4 GABRA3 5 GM6644 6 GM6614 7 AKR1B3 8 BCL6 Ion and chloride transport Gamma-aminobutyric acid signaling pathway Synaptic transmission Sorbitol biosynthetic process Steroid metabolic process S-activated protein kinase signaling cascade Drug transmembrane transport, drug transport methotrexate transport Sorbitol biosynthetic process Steroid metabolic process Stress-activated protein kinase signaling cascade oxidation-reduction process Protein import into nucleus Translocation, cell morphogenesis Negative regulation of type 2 immune response transcription Rho protein signal transduction, spermatogenesis, protein localization, B cell differentiation, negative regulation of cell growth 0.8 0.2 0.3 0.3 1.4 0.3 0.3 0.3 0.5 0.3 9 TREML4 Signal transduction 0.7 0.3 10 CRELD2 B process 2.4 0.3 11 DIO1 Metabolic process Hormone biosynthetic process Oxidation-reduction process 1.6 0.3-29 -
FMO family SOA (1 day) Fold-increase by PBE MOA (7 days) FMO1 NC NC FMO2 1.5 3.8 FMO3 5.1 17.6 FMO4 1.3 3.1 FMO5 NC NC - 30 -
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Number Number of Analysis of design Cutoff Regulation Number of of GO Pathway Significant Gene information Information DOWN 1264 901 282 1.3 UP 1184 925 349 DOWN 292 184 75 1.5 UP 328 226 93 Control vs PBE-100 DOWN 61 31 14 2 UP 45 36 18 DOWN 22 1 0 3 UP 9 8 7 NO Gene Symbol Accession Number Liver Control Liver PBE-100 Average Fold Change 1 Fmo3 NM_008030 7.0 11.2 17.6 2 Mt2 NM_008630 10.2 13.7 11.3 3 Cyp46a1 NM_010010 7.8 10.9 8.8 4 Corin NM_016869 6.6 9.2 5.8 5 Mt1 NM_013602 9.5 11.7 4.6 6 Akr1e1 NM_018859 7.8 9.9 4.2 7 Plk3 NM_013807 9.1 11.1 3.8-43 -
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제 3 절 MT 유전자의발현과효능 β β - 60 -
Control Pb group Cd group Group Pb(or Cd) PBG ZYM Pb o Pb-PBG o o Pb-ZYM o o Cd o Cd-PBG o o Cd-ZYM o o - 61 -
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항목내용비고게재연도 2016 논문명 저자명 Induction of Flavin-containing Monooxygenase in Mice by Oral Administration of Phellinus baumii (Agaricomycetes) Extract Batbayar Sainkhuu, Beom Su Park, Ha Won Kim 학술지명 International Journal of Medicinal Mushrooms IJMM Vol.(No.) 18(9) 국내외구분국외 ( 미국 ) SCI 구분 SCIE 기타페이지 : 793-806 - 65 -
항목내용비고 게재연도 2017 논문명 Activation of NADPH Oxidase by Beta-Glucan of Phellinus baumii Pilat (Hymenochaetaceae, Higher Basidiomycetes) in RAW264.7 Cells 저자명 학술지명 Vol.(No.) 19(11) 국내외구분국외 ( 미국 ) Soo Kyung Sung, Sainkhuu Batbayar, Dong Hee Lee and Ha Won Kim International Journal of Medicinal Mushrooms (IJMM) 인쇄중 ( 게재확정 ) SCI 구분 기타 SCIE 코드번호 C-06-02 - 66 -
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제 5 장연구결과의활용계획 코드번호 D-07 3. 중금속배출타깃기술 체내중금속배출에대한안전한기술이많지않으므로상황버섯을비롯한다양한버섯류의베타 - 글 루칸에대하여중금속배출에관한과제를추진예정임. - 78 -
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주 의 1. 이보고서는농림축산식품부에서시행한농생명산업기술개발 사업의연구보고서입니다. 2. 이보고서내용을발표하는때에는반드시농림축산식품부에 서시행한농생명산업기술개발사업의연구결과임을밝혀야합 니다. 3. 국가과학기술기밀유지에필요한내용은대외적으로발표또는 공개하여서는아니됩니다. - 89 -