舞茸生物活性代謝產物之醱酵生產與其功能評估

Abstract

本研究利用深部培養法生產 Grifola frondosa NTUS 之生物活性代謝產物，並進行生物活性代謝產物之分離與鑑定及探討其抗腫瘤機制。同時以生物活性代謝產物作為活性指標，進行最適醱酵培養基及最適醱酵條件之探討，其主要結果分述如下： G. frondosa NTUS 分別以種菌培養基及β-葡聚醣生產培養基於氣舉式醱酵槽中培養14天，離心後菌絲體及上澄液分別以乙酸乙酯進行萃取，共可得8個區分物 (EFMS、EFFS、EFMG、EFFG、WFMS、WFFS、WFMG、WFFG)，以MTT法進行肝癌細胞生長抑制測試，發現深部培養液之乙酸乙酯萃取物皆具有抑制肝癌細胞生長活性，其中以種菌培養基培養所得上澄液之乙酸乙酯萃取物EFFS活性最高，進一步將EFFS進行不同人類癌細胞之生長抑制測試，發現EFFS同時對人類肺癌細胞 (CL 1-1)、子宮頸癌細胞 (HeLa)、肝癌細胞 (Hep G2、Hep 3B) 均具有明顯抑制效果，其IC50分別為78.4，52.7，77.6 及 71.0 μg/mL，而EFFS對正常細胞MRC-5抑制效果較不顯著，其IC50為223.3 μg/mL。 Hep 3B 以EFFS處理後，其細胞核型態以DAPI螢光染色及DNA電泳進行分析，當以200 μg/mL EFFS處理Hep 3B 36小時，可誘導Hep 3B細胞染色體凝聚及DNA片段化。由流式細胞儀分析結果可知， EFFS 可使Hep 3B 細胞發生S期停滯現象，並有sub-G1 波峰出現，推測EFFS藉由細胞凋亡機制導致人類肝癌細胞死亡。 以矽膠管柱層析及半製備式HPLC進行EFFS 區分，可得一主要腫瘤細胞生長抑制活性化合物EFFS-HE-5-5，經UV、GC-MS及1H-NMR分析結果可知EFFS-HE-5-5為o-orsellinaldehyde (2,4-dihydroxy-6-methylbenzaldehyde)。 自Sigma-Aldrich購買之 o-orsellinaldehyde 標準品其HPLC層析圖譜與Hep 3B 細胞生長抑制活性與分離所得 EFFS-HE-5-5 一致。o-Orsellinaldehyde對人類肝癌細胞Hep 3B 及肺纖維母細胞細胞MRC-5存活率之IC50分別為3.6及33.1 μg/mL，顯示o-orsellinaldehyde 對Hep 3B 及MRC-5具有選擇性的細胞毒性。Hep 3B 以o-orsellinaldehyde處理後，其細胞核型態以DAPI螢光染色及DNA電泳進行分析，當以10 μg/mL o-orsellinaldehyde處理Hep 3B 48小時，可誘導Hep 3B細胞染色體凝聚及DNA片段化。由流式細胞儀分析結果可知，o-orsellinaldehyde 可使Hep 3B 細胞發生S期停滯現象，並有sub-G1 波峰出現，推測o-orsellinaldehyde藉由細胞凋亡機制導致人類肝癌細胞死亡。 經由西方墨點法分析，o-orsellinaldehyde 會對Hep 3B 細胞週期調控蛋白質cyclin A及p21進行上升調節，導致細胞週期停滯於S期。經2-DE分析及LC/MS/MS鑑定，推測o-orsellinaldehyde 可對訊息傳遞路徑蛋白質 (protein disulfide isomerase、F-actin capping protein alpha-1 subunit、stathmin)、轉錄、轉譯作用調控蛋白質 (cytokine induced protein 29、alternitive、ATP synthase) 進行上升調節，對訊息傳遞路徑蛋白質 (annexin V、chaperonin containing TCP1)、轉錄、轉譯作用調控蛋白質 (peptidyl-prolyl cis-trans isomerase B precursor) 及細胞骨骼系統 (WD repeated-containing protein 1 isoform 1、acetoacetyl-CoA thiolase) 進行下降調節，因而抑制Hep 3B 細胞生長並誘發細胞凋亡。 以Hinton 氏三角瓶探討G. frondosa NTUS 生產生物活性代謝產物之最適培養基組成，得知其最適培養基如下：0.6% malt extract，0.24% yeast extract，0.1% MgSO4•7H2O，3% glucose，pH 4.5。以此培養基於裝液量100 mL 之500 mL Hinton氏三角瓶於25℃下震盪培養9天，可得到18.2 g/L菌體量，2.0 g/L 胞外多醣及13.8 mg/L o-orsellinaldehyde，醱酵上澄液乙酸乙酯萃取物對Hep 3B 細胞之IC50值為72.3 μg/mL，且此萃取物亦會藉由細胞凋亡及細胞週期S期停滯抑制Hep 3B 細胞生長。此外亦從乙酸乙酯萃取物分離出另一活性代謝產物，經GC-MS分析，推測此化合物為4,5-dimethyl-1,3-benzenediol。 以β-葡聚醣生產培養基在500 mL Hinton 氏三角瓶進行固定化G. frondosa NTUS 菌絲體之胞外多醣生產，於25℃下培養120小時可獲得胞外多醣3.1 g/L，以固定化菌絲體進行連續批次培養，培養至第六批次胞外多醣之產量由原本3.1 g/L降至 1.5 g/L。以三公升固定化生物反應器進行胞外多醣生產，培養至第五天有最大胞外多醣產量2.5 g/L。以含生物活性代謝產物最適化培養基之五公升固定化生物反應器進行生物活性代謝產物之生產，於通氣量2 L/min、攪拌速率 150 rpm及 pH 4.5 培養3天可得8.7 mg/L o-orsellinaldehyde，培養至第9天可得0.8 g/L之胞外多醣及7.2 mg/L o-orsellinaldehyde。

The purpose of this study was to isolate and identify the bioactive metabolites from submerged culture of Grifola frondosa NTUS and to study the mechanism of its antitumor activity. The identified metabolite was used as biomarker to optimize the medium composition and fermentation conditions of G. frondosa NTUS. G. frondosa NTUS was cultured in seed culture medium and β-glucan production medium, respectively, in airlift fermentor for 14 days. After centrifugation, mycelium and supernatant were extracted by ethyl acetate as eight fractions (EFMS, EFFS, EFMG, EFFG, WFMS, WFFS, WFMG, WFFG). Inhibition of Hep 3B cell proliferation was measured by MTT assay. All of the ethyl acetate extracts contained inhibitory activity against Hep 3B cells growth. Ethyl acetate fraction of filtrate fermented in seed culture medium (EFFS) showed the highest inhibitory activity. The IC50 of EFFS for cytotoxicity against human carcinoma cells (Hep 3B, Hep G2, HeLa, CL1-1) and normal human lung fibroblast MRC-5 was 78.4, 52.7, 77.6, 71.0 and 233.3 μg/mL, respectively. The nuclear morphology of EFFS-treated Hep 3B cells was analyzed using the DNA-binding dye DAPI and agarose electrophoresis assay. Hep 3B cells exposed to 200 μg/mL EFFS for 36 h exhibited condensed chromatin and fragmented nuclei. EFFS induced S-phase arrest and the presence of a significant sub-G1 peak by flow cytometry suggest that EFFS might mediate its cytotoxicity through apoptosis. EFFS was fractionated by silica gel chromatography and semi-preparative HPLC. A main tumoricidal active compound, EFFS-HE-5-5, was obtained. According to UV, GC-MS and H1NMR data, EFFS-HE-5-5 was identified as o-orsellinaldehyde (2,4-dihydroxy-6-methylbenzaldehyde). The commercial standard of o-orsellinaldehyde purchased from Sigma-Aldrich also showed the same growth inhibitory activity against Hep 3B cells with EFFS-HE-5-5. The IC50 of o-orsellinaldehyde based on the cell viability of Hep 3B and MRC-5 cells was 3.6 and 33.1 μg/mL, respectively. Thus, o-orsellinaldehyde showed a selective cytotoxic effect against Hep 3B and MRC-5 cells. The nuclear morphology of o-orsellinaldehyde-treated Hep 3B cells was analyzed using the DNA-binding dye DAPI and agarose electrophoresis assay. Hep 3B cells exposed to 10 μg/mL o-orsellinaldehyde for 48 h exhibited condensed chromatin and fragmented nuclei. o-Orsellinaldehyde induced S-phase arrest and the presence of a significant sub-G1 peak by flow cytometry suggest that o-orsellinaldehyde might mediate its cytotoxicity through apoptosis. Accroding to western blot assay, o-orsellinaldehyde might induce Hep 3B to arrest in S phase via up-regulating expression of cyclin A and p21. 2-DE and LC/MS/MS analyses suggested that o-orsellinaldehyde might inhibit Hep 3B cell growth and induce apoptosis via up-regulating expression of proteins involved in signal transduction (protein disulfide isomerase, F-actin capping protein alpha-1 subunit and stathmin), transcriptional and translational control (cytokine induced protein 29, alternative and ATP synthase), and via down-regulating expression of proteins involved in signal transduction (annexin V and chaperonin containing TCP1), transcriptional and translational control (peptidyl-prolyl cis-trans isomerase B precursor), and cytoskeleton organization (WD repeated-containing protein 1 isoform 1 and acetoacetyl-CoA thiolase). The optimum medium for production of bioactive metabolites by G. frondosa NTUS was investigated in Hinton flask. It was found that the optimum medium was composed of 0.6% malt extract, 0.24% yeast extract, 0.1% MgSO4•7H2O, 3% glucose and pH 4.5. After cultured at 25℃ in a Hinton flask for 9 days, the biomass, extracellular polysaccharides, and o-orsellinaldehyde obtained were 18.2 g/L, 2.0 g/L, and 13.8 mg/L, respectively. The IC50 of ethyl acetate extracts of culture supernatant for cytotoxicity against Hep 3B was 72.3 μg/mL and it also mediated its inhibitory activity against Hep 3B cells through S-phase arrest and apoptosis. At the same time, another active compound was isolated from ethyl acetate extracts and it was predicted as 4,5-dimethyl-1,3- benzenediol after GC-MS analysis. Production of extracellular polysaccharides by immobilized G. frondosa NTUS was performed in 500 mL Hinton flask which contained 100 mL β-glucan production medium. After cultured at 25℃ in a Hinton flask for 120 h, extracellular polysaccharides obtained was 3.1 g/L. After six repeated batch, extracellular polysaccharides obtained was 1.5 g/L. The production of extracellular polysaccharides was also performed in a 3-L loofa sponge immobilized bioreactor. The highest production of extracellular polysaccharides obtained was 2.5 g/L after 120 h cultivation. The production of bioactive metabolites in a 5-L loofa sponge immobilized bioreactor loaded optimum medium was performed under the conditions as follows: aeration rate, 2 L/min; agitation speed, 150 rpm and pH 4.5 at 25 ℃. After cultured for 3 days, o-orsellinaldehyde obtained was 8.7 mg/L. After cultured for 9 days, extracellular polysaccharides and o-orsellinaldehyde obtained were 0.8 g/L and 7.2 mg/L, respectively.