探討牛樟芝活性成分4-acetylantroquinonol B 之生合成 途徑以改善其液態發酵之方法

Abstract

牛樟芝(Antrodia cinnamomea)是台灣特有真菌，其代謝產物具有許多生物活性。由於牛樟芝子實體生長速度緩慢，目前產業界常藉由液態培養的方式培養菌絲體以取得特定的活性成分。牛樟芝具有藥理活性功效的成分，主要可分為多醣體、三萜類、二萜類、馬來酸與琥珀酸衍生物及泛醌類，而其中泛醌類化合物，包括antroquinonol及其衍生物antroquinonol B及4-acetylantroquinonol B對多株人類癌細胞有很強的毒殺作用，同時具有不錯的抗發炎活性，其構造與活性皆有專利申請保護。本實驗室過去之研究從牛樟芝菌絲體乙醇萃出物分離出4-acetylantroquinonol B，並證實4-acetylantroquinonol B為目前牛樟芝活性成分中抑制肝癌細胞HepG2生長效果最佳者，並發現4-acetylantroquinonol B會增加肝癌細胞HepG2 p53、p21、p27的表現，進而讓細胞週期停滯於G0/G1期，使其無法生長，IC50為0.1 μg/mL。因此，本研究希望透過了解4-acetylantroquinonol B的結構特徵，參考ubiquinone的生合成途徑，利用添加前軀物及其他輔助因子來提升其產量。解析4-acetylantroquinonol B的結構後將其分為三部分: (1) quinone環，(2) 異戊二烯碳鏈及(3) γ-lactone，再由生合成途徑來推測各部分可能的前軀物。實驗結果證實，添加0.01% 4-hydroxybenzoic acid (3.7 mg/g dried mycelium)、0.01% CoQ0 (5.6 mg/g dried mycelium)及0.01% CoQ10 (4.8 mg/g dried mycelium)皆可能因增加quinone環的原料而提升產量，而添加0.1% olive oil (3.0 mg/g dried mycelium)、0.1% oleic acid (3.6 mg/g dried mycelium)，推測可以增加異戊二烯碳鏈及γ-lactone的原料而增加產量。並且，添加0.1% MgSO4∙7H2O (6.5 mg/g dried mycelium)，可能是藉由增加生合成途徑所需酵素的輔助因子，亦可顯著提升4-acetylantroquinonol B產量。又考量牛樟芝對牛樟木宿主的專一性，也透過添加牛樟木中所含的精油成分，發現0.01% α-terpeniol (2.7 mg/g dried mycelium)及0.01% geraniol (1.7 mg/g dried mycelium)亦可增加4-acetylantroquinonol B產量。綜合上述實驗結果，本研究也針對不同結構部分的前軀物合併添加，發現添加0.01% 4-hydroxybenzoic acid及 0.01% CoQ0 (27.8 mg/g dried mycelium)、0.01% 4-hydroxybenzoic acid及0.1% oleic acid (5.1 mg/g dried mycelium)和0.01% 4-hydroxybenzoic acid及0.01% geraniol (3.9 mg/g dried mycelium)皆可以比個別添加作為前軀物有更佳的提升4-acetylantroquinonol B產量的效果。

Antrodia cinnamomea (AC) is a treasured endemic Taiwanese medicinal mushroom. Its secondary metabolites possess many biological functions. Due to the fact that the fruiting body of AC grows very slow, the cultivation of mycelium by submerged fermentation is now a common practice for the production of bioactive compounds. The major bioactive compounds of AC are polysaccharides, triterpenes, diterpenes, succinic acid and maleic acid derivatives and ubiquinone. Previous study of our lab has purified 4-Acetylantroquinonol B (4-AAQB), an ubiquinone derivate, and proved that it is the major antiproliferative compound for hepatoma cells. 4-AAQB can mediate cell cycle arrest via decreases of CDK2 and CDK4 and increase p53, p21 and p27, and this lead to the proportion of cells in the G0/G1 phase increased, and that in the S phase decreased. The IC50 value of 4-acetylantroquinonol B toward HepG2 cells is 0.1 μg/mL. The objective of this study was to increase the production of 4-AAQB. Since the 4-AAQB is a kind of ubiquinone derivatives, this study referred the biosynthesis pathway and biological function of ubiquinone, and based on the three parts of the chemical structures of 4-acetylantroquinonol B, including Quinone ring, isoprenoid chain and γ-lactone, to investigate several possible precursors for increasing production of 4-acetylantroquinonol B. The results showed that 0.01% 4-hydroxybenzoic acid (3.7 mg/g dried mycelium), 0.01% CoQ0 (5.6 mg/g dried mycelium) and 0.01% CoQ10 (4.8 mg/g dried mycelium) supplementation could increase the 4-AAQB production possibly throughout the increase raw material needed for the construction of quinone ring. The 0.1% olive oil (3.0 mg/g dried mycelium), 0.1% oleic acid (3.6 mg/g dried mycelium) supplementation could increase the 4-AAQB production possibly by increasing the materials needed for biosynthesis of isoprenoid chain and γ-lactone. And from the cofactor of enzyme in the biosynthesis pathway, 0.1% MgSO4∙7H2O (6.5 mg/g dried mycelium) supplementation could also increase the amount of 4-acetylantroquinonol B. From the aforementioned results we can deduce that the biosynthesis between 4-acetylantroquinonol B and ubiquinone may be similar or closely related. Since AC can only grow on Cinnamomum kanehirai Hay, I chose two essential oils , α-terpeniol and geraniol from the host tree, and found that 0.01% α-terpeniol (2.7 mg/g dried mycelium) and geraniol (1.7 mg/g dried mycelium) supplementation could increase the synthesis of 4-AAQB. Finally, I combined the precursors from quinone ring and isoprenoid chain, and found that combined supplementation of 0.01% 4-hydroxybenzoic acid and 0.01% CoQ0 (27.8 mg/g dried mycelium)、0.01% 4-hydroxybenzoic acid and 0.1% oleic acid (5.1 mg/g dried mycelium) or 0.01% 4-hydroxybenzoic acid and 0.01% geraniol (3.9 mg/g dried mycelium), all had synergistic effect on the production of 4-acetylantroquinonol B.