牛樟芝菌絲體中antroquinonol與4-acetylantroquinonol B 生合成路徑之研究

Abstract

牛樟芝是台灣原生真菌物種，久被用為抗癌及護肝的營養補充品。牛樟芝中含有多種特有的活性成分，但其利用受限於有限的野外資源及緩慢的生長速度。2007年在牛樟芝菌絲體中發現的antroquinonol (AQ)，目前在臺灣及美國進行非小細胞肺癌的第二期新藥的人體臨床試驗。僅能在牛樟芝菌絲體中發現的4-acetylantroquinonol B (4-AAQB)，與AQ有與coenzyme Q (CoQ)類似的化學結構。基於AQ與4-AAQB的benzoquinone環形結構可能源自shikimate pathway與polyketide pathway的假設，本實驗以穩定同位素代謝標定法，使用高效液相層析串聯高解析度軌道阱式質譜儀執行相關分析工作。結果顯示AQ與4-AAQB的生合成路徑與CoQ類似，由acetyl-CoA與 malonyl-CoA經polyketide pathway生合成之orsellinic acid (OA)，是coenzyme Q3 (CoQ3)、AQ與4-AAQB的benzoquinone環形結構前驅物。此外，由shikimate pathway生合成之4-hydroxybenzoic acid (4-HBA)為CoQ3及AQ的環形結構前驅物，但並非4-AAQB的環形結構前驅物。牛樟芝利用內生的4-HBA生合成AQ，芳香族胺基酸tyrosine與phenylalanine在shikimate pathway被glyphosate阻斷時，亦可轉化為4-HBA進而生合成AQ。4-AAQB的benzoquinone環形結構僅能由來自polyketide pathway生合成之OA所構成，但AQ的benzoquinone環形結構可由來自shikimate pathway生合成之4-HBA與polyketide pathway生合成之OA所構成。本文最後並提出由實驗證據所彙整後所推測之牛樟芝中AQ與4-AAQB生合成路徑的前驅物-產物關係圖。

Antrodia cinnamomea, an endemic fungus species of Taiwan, has long been used as a luxurious dietary supplement to enhance liver functions and as a remedy for various cancers. They produce a diverse array of bioactive secondary metabolites, many of which are unique to the organisms. Their potential applications, however, are limited by their finite sources and the slow-growing nature of the organisms in both laboratory and environmental conditions. Antroquinonol (AQ), identified from the mycelium of A. cinnamomea, is currently in phase II clinical trials in the USA and Taiwan for the treatment of non-small-cell lung cancer. AQ and 4-acetylantroquinonol B (4-AAQB), isolated from the submerged fermentation mycelium of Antrodia cinnamomea, have similar chemical backbone to that of coenzyme Q (CoQ). Based on the postulation that biosynthesis of both AQ and 4-AAQB in A. cinnamomea starts from polyketide pathway and shikimate pathway, the strategy of metabolic labeling with stable isotopes was applied, and analyzed by the ultrahigh-performance liquid chromatography coupled to a high-resolution quadrupole orbital-trap mass spectrometry. We found that AQ and 4-AAQB follow the similar biosynthetic sequences as that of CoQ. The orsellinic acid, formed from acetyl-CoA and malonyl-CoA via polyketide pathway, was found to be a novel benzoquinone ring precursor for coenzyme Q3 (CoQ3), AQ and 4-AAQB. Besides, we also confirmed that 4-hydroxybenzoic acid (4-HBA), formed via shikimate pathway, serves as the ring precursor of AQ but not serves as that of 4-AAQB. A. cinnamomea preferentially utilizes endogenous 4-HBA via shikimate pathway for AQ biosynthesis. Exogenous tyrosine and phenylalanine can be utilized for AQ biosynthesis when shikimate pathway is blocked by glyphosate. The benzoquinone ring of 4-AAQB synthesizes only via polyketide pathway, and that of AQ synthesizes via both polyketide pathway and shikimate pathway. The precursor-products relationships of AQ and 4-AAQB in A. cinnamomea are proposed based on the experimental findings.