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標題: 白芷不同生長時期活性物質含量及相關基因選殖之研究
Analysis of Physiologically Activated Compounds and Cloning of Related Genes Involving Developmental Stages of Angelica dahurica
作者: 羅淑卿
Lo, Shu-Chin
關鍵字: 白芷;Angelica dahurica;香豆素;美白;coumarins;skin-whitening
出版社: 生物科技學研究所
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白芷具有美白潤膚之功效。已知其中的一個美白成分為氫氧基化佛手柑內酯 (8-hydroxybergapten),由苯基丙烷類代謝路徑 (phenylpropanoids) 合成。為選殖及探討參與合成8-hydroxybergapten之酵素基因特性,利用5´-與3´-RACE-PCR對白芷根Cinnamate 4-hydroxylase (AdC4H) 及bergaptol -O-methyltransferase (AdBMT) cDNA片段進行聚合酶連鎖增幅反應取得序列全長。含有AdBMT基因之大腸桿菌可簡便有效率的轉化佛手柑內醇 (bergaptol) 成大量的佛手柑內酯 (bergapten),可成為有潛力的生物反應器。將AdC4H可編譯區cDNA建構在pYES2/NT,另外選殖並建構阿拉伯芥cytochrome P450 reductase ATR1全長於pYES3/CT,以缺乏色胺酸及尿嘧啶之培養基篩選,取得併存二載體 (pYES2/NT-C4H及pYES3/CT-ATR1) 之轉形酵母菌 (Saccharomyces cerevisiae),可提高AdC4H催化效率。於200 μM trans-cinnamic acid培養基液中培養含AdC4H基因之酵母菌,以30℃培養24小時,即可得到122 μM ρ-coumaric acid。使用UPLC管柱為ethylene bridged hybrid (BEH) C18,在酸化水 (pH 3.0) 沖提下變化氰甲烷 (acetoniltrile) 濃度由15%至95%使成梯度流洗,可在12分鐘之內同時有效率的分離並定量七種香豆素。呋喃香豆素psoralen、xanthotoxin、bergapten及8-hydroxybergapten皆可在種植9個月的白芷根部偵測到,其中8-hydroxybergapten含量皆高過其它呋喃香豆素10餘倍。種植3個月及6個月之白芷莖抑制酪胺酸酶能力為最佳。白芷AdC4H mRNA主要表現於種植9個月之根、莖及葉。AdBMT mRNA表現於各器官,以種植6個月之莖及9個月之葉比較多。白芷莖部之高抑制酪胺酸酶能力,未來可萃取白芷莖香豆素應用於化妝品工業。

Bai Zhi (Angelica dahurica) has long been used as a face cream for skin-whitening purposes. One of the known skin-whitening components, 8-hydroxybergapten is synthesized from phenylpropanoids pathway. To clone and characterize of related genes involved in 8-hydroxybergapten synthesis, the tandem 5´- and 3´-rapid amplification of cDNA ends via polymerase chain reaction was used to obtain the full-length cinnamate 4-hydroxylase (AdC4H) and bergaptol-O-methyltransferase (AdBMT) cDNA sequences from Bai Zhi root. A simple and efficient production of bergapten in the E. coli culture overexpressing AdBMT was performed. With the supply of bergaptol in the medium, E. coli cells can be used as a potential bioreactor to produce bergapten. The full length of AdC4H coding region was introduced into pYES2/NT vector. Besides, the full length of cytochrome P450 reductase ATR1 cloned from Arabidopsis thaliana was introduced into pYES3/CT vector. The transformed yeast cell (Saccharomyces cerevisiae) harboring two different plasmids (pYES2/NT-C4H and pYES3/CT-ATR1) were obtained by growing in the medium without containing tryptophan and uracil. The AdC4H together with ATR1 overexpression showed highly efficient catalysis of 4-hydroxylation of cinnamate. The amount of 122 μM ρ-coumaric acid could be obtained from yeast harboring two different plasmids that cultured in medium containing 200 μΜ trans-cinnamic acid at 30℃ for 24 hours. An improved ultra performance liquid chromatography method was developed to simultaneously quantify seven coumarins isolated from various Bai Zhi organs. It can be achieved within 12 min using ethylene bridged hybrid (BEH) C18 column and a gradient elution system at acetonitrile concentrations varying from 15 to 95% (v/v) in acidic water (pH 3.0) eluent. The furanocoumarins such as psoralen、xanthotoxin、bergapten and 8-hydroxybergapten were detected in 9 month-old Bai Zhi roots, in which the content of 8-hydroxybergapten was 10-fold higher than other coumarins. Nevertheless, stems from 3 and 6 month-old Bai Zhi exhibited the highest inhibitory ability on tyrosinase. RNA blot analysis indicated that AdC4H mRNAs was predominantly expressed in 9 month-old Bai Zhi roots, stems, and leaf organs, whereas AdBMT mRNAs was expressed in every organ of the three different organs with higher expression in 6 month-old Bai Zhi stem and 9 month-old Bai Zhi leaf. In the future, the Bai Zhi stem coumarins with high inhibitory effect on tyrosinase could be further extracted and used in cosmetic industry.
其他識別: U0005-0908201217283900
Appears in Collections:生物科技學研究所

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