大型樟芝發酵液對肝癌細胞Hep G2生長抑制之探討

Abstract

樟芝是台灣傳統用藥，具有抑制肝癌細胞Hep G2的活性，本研究是利用液態深層培養，以250 mL搖瓶、6.6公升、500公升及5噸發酵槽依序將樟芝發酵製程放大。首先以搖瓶探討培養基發酵條件，以抑制Hep G2生長作為篩選依據，結果發現最佳的碳源與氮源分別為葡萄糖與麥芽抽出物，培養基的初始 pH 值為5，發酵溫度22℃。將此發酵條件用於6.6公升、500公升及5噸發酵槽，持續監測Hep G2生長作為篩選依據，結果發現以6.6公升發酵四週時，對Hep G2的濃度 IC50 為3.8 μg / mL，將發酵延長至八週並無法降低 IC50。在發酵過程的監測項目中，以HPLC發現樟芝菌絲體中含有的麥角固醇隨著發酵時間而持續增加，發酵濾液的還原糖與菌絲體乙醇萃取物有很高的指數相關性(相關係數0.89)，所以菌絲體的麥角固醇和發酵濾液的還原糖含量對於樟芝抑制Hep G2的活性具有發酵指標的意義，可以應用於即時監測發酵製程。

The scale up of submerged cultivation of Antrodia cinnamomea was carried out for manufacturing the fermentation product with anti-hepatoma activity. The fermentation process was optimized for different parameters at shake flask level to get products with high inhibition potency against Hep G2 hepatoma cells. Glucose and malt extract were found to be the best carbon and nitrogen source, respectively. The initial pH of 5.0 and an operating temperature of 22℃ were the best for a product with lowest IC50 value. Scale up of the fermentation process was achieved from 250 mL shake flask to 5 liter, 500 liter and 5 Ton fermenters. It was found that the cultivation time could be decreased from 8 weeks to 4 weeks when the scale of fermentation increased from 5 liter to 5 ton. HPLC analyses of mycelium extracts of different fermentation scales and cultivation times indicated that ergosterol could serve as a marker for the anti-hepatoma potency. It was further observed that reducing sugar content of the fermentation broth were correlated exponentially with the IC50 of the ethanolic extract of mycelium for hepatoma cells, and might be used as a indicator for monitoring the fermentation process.