| Project/Area Number |
12672060
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Chemical pharmacy
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| Research Institution | Kitasato University |
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Principal Investigator |
ASADA Yoshihisa Kitasato Univ., School of Pharm. Sci., Associate Professor, 薬学部, 助教授 (90118779)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIKAWA Takafumi Kitasato Univ., School of Pharm. Sci., Professor, 薬学部, 教授 (80050540)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2001: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2000: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | forskolin / biosynthesis / non-mevalonate pathway / Coleus forskohlii / hairy root / diterpene / constituent / Labiata / mevalonate pathway |
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| Research Abstract |
1. [1-^<13>C]glucose was administered to the Coleus forskohlii hairy roots cultured for 2 weeks in B5 liquid medium. Then, the hairy roots were cultured for 2 weeks and harvested. The MeOH extract was extracted with EtOAc according to a conventional method. The EtOAc extract was subjected to silica gel chromatography and then purified by repeated reversed-phase HPLC to give ^<13>C-labeled forskolin and its analogs. The incorporation patterns of [1-^<13>C] glucose into these compounds demonstrated that diterpenes in the C. forskohlii hairy root were biosynthesized via non-mevalonate pathway. Furthermore, labeling pattern of betulinic acid derived from [1-^<13>C] glucose was consistent with that biosynthesizing via mevalonate pathway. It became clear that mevalonate and non-mevalonate pathways are working in C. forskohlii hairy roots.
2. For the isolation of intermediates in forskolin biosynthesis, we investigated the constituents of C. forskohlii hairy roots to give 17 diterpenes containing 4 new compounds. On the basis of the structures of isolated compounds, the biosynthetic pathway of forskolin was proposed.
3. The cloning of diterpene cyclase was performed by RT-PCR on the basis of the amino acid alignment of copalyl diphosphate synthase (CPS) which cDNA has already been cloned. Consequently, the clone which has similarity with CPS was not obtained.
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