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Studies on Antioxidant and Antitumor Properties of Antrodia camphorata in Submerged Culture
|關鍵字:||Antrodia camphorata in Submerged Culture;樟芝深層培養液;Antioxidant;Antitumor;Hepatoma cells;Apoptosis;抗氧化;抗腫瘤;肝癌細胞;細胞凋亡||出版社:||食品科學系||摘要:||
本論文乃欲探討樟芝深層培養(Antrodia camphora of submerged culture; ACSC)萃取物的抗氧化及抗腫瘤特性。乃將樟芝深層培養萃取物分為濾液乾燥物(DMF)和不同溶劑之菌絲體萃取物與培養基乾燥物(DMCM)進行抗氧化及自由基清除能力之探討，並分析各萃取物中可能之抗氧化成分及含量，以期找出具有抗氧化能力之活性成分。DMF顯示具有最佳之抑制脂質過氧化能力，且抑制能力隨著濃度增加而有上升的趨勢。在0.2 mg/ml之濃度下，其抑制能力約相等於同濃度的BHA (60%)。菌絲體萃取物中，除了正己烷萃取物具有較弱的抗氧化力外，其他萃取物在0.2 mg/ml之濃度下，亦具有>40%的抑制脂質過氧化能力。DMF及菌絲體水萃取物顯示有較強的自由基清除能力，且二者之抗氧化活性與其含有的總多酚類、粗三類含量及粗多醣體中所含有之蛋白質/多醣體比率有關。在不同的測試系統中，DMCM的抗氧化能力均不如DMF，顯示DMF中之主要抗氧化成分，應來自於菌絲體的二次代謝產物。由此可知，DMF可能具有開發為預防自由基相關疾病之化學預防療劑的潛力。
此外本論文也探討ACSC之發酵濾液乾燥物(DMF)是否具有保護H2O2誘導HepG2產生細胞毒性及SD大鼠產生肝毒性的效果，並探討其具有保護效果的可能機制。初步的試驗結果顯示，DMF及其粗三類在AAPH/linoleic acid系統中具有濃度效應地抑制脂質過氧化能力。0.10 mg/ml之DMF與HepG2預培養4 h後，可顯著降低H2O2 (100 M)處理1 h所誘導之MDA形成(p<0.05)。在活體試驗結果顯示，先以DMF (0.25及0.5 g/kg) 連續餵食SD大鼠五天，能顯著降低單一劑注射40%之CCl4 (0.1 ml /100g b.w., i.p.)所誘導肝臟血清之酵素指標(ALT及AST)及抑制脂質過氧化 (p<0.05)。在肝臟組織病理切片中發現，DMF能降低CCl4對大鼠引發之嗜中性白血球浸潤 (neutrophil infiltration)、水泡性腫大(hydropic swelling)及壞死(necrosis)等肝損傷現象。除此外，預先餵食大鼠DMF後，亦可顯著提升CCl4誘導肝組織之GSH-dependent enzymes及GSH/GSSG ratio的下降(p<0.01)。基於上述結果，我們推測DMF具有提升大鼠肝組織GSH-dependent enzymes的活性(Glutathione peroxidase, Glutathione reductaser及Glutathione S-transferase)以維持肝組織之GSH/GSSH ratio恆定比率，及直接清除CCl4代謝產生的自由基。因此具有預防活體內氧化傷害的潛力。
本論文亦探討樟芝深層培養菌絲體甲醇萃取物(MEM)之抑制癌細胞存活能力，並評估其毒殺作用機制，以期找出可能的調控路徑。研究結果顯示，以不同濃度之MEM處理人類肝癌細胞株HepG2及Hep3B 48 h，其IC50分別為49.5及62.7g/ml，其抑制癌細胞存活能力約為野生樟芝子實體甲醇萃取物(MEAF)之1/5；且在小於100 g/ml之濃度下，對Chang liver cells及大鼠初代肝細胞並不具有明顯的毒性效果。由細胞形態變化、DNA斷鏈分析、細胞週期變化的結果可知，MEM對肝癌細胞之毒殺路徑是經由誘導凋亡而非壞死作用。由細胞週期分析發現，MEM誘導HepG2凋亡的路徑與停止細胞週期在G1 期有關，而Hep3B則無此趨勢。以MEM (100 g/ml)與HepG2 (wild-type p53)及Hep3B (delete p53)培養72 h後，凋亡細胞數分別為98.3及39.5%，因此推測p53可能是MEM調控HepG2凋亡的敏感性大於Hep3B的一個重要因子。MEM誘導HepG2細胞凋亡之主要路徑應是藉由停止細胞週期於G1期，及增加Fas表現以活化caspase-8及-3，進而造成細胞凋亡。
最後本論文將樟芝深層培養菌絲體甲醇萃取物(MEM)以高效能液相層析(high performance liquid chromatography; HPLC)分離區分後，以抑制肝癌細胞(Hep3B)存活能力來評估各區分物之抗癌效果，並選擇含量較多及抗癌能力較佳之區分物進行再純化及鑑定。MEM經分析型管柱以HPLC分離得六個區分物(A~F)，由積分面積得知，以C區分物含量最多(40%)，其次為B區分物(16%)及E區分物(13%)，其餘區分物約4-8%。B、C、D、E與Hep3B培養24 h後，其IC50約為26-45 g/ml，A及F則無明顯的抑制Hep3B存活能力。取C區分物以製備型管柱進行HPLC分離及TLC純化後，由紫外-可見光(UV-Vis)、紅外光(IR)、質譜(Mass)及核磁共振(1H-NMR, 13C-NMR)光譜分析結果，鑑定分子式為C16H22O3，分子量為262.2的化合物。
This study evaluated antioxidant and Anti-tumor properties of extracts from Antrodia camphorata in submerged culture (ACSC). Chapter 2 focuses on the studies of biologically active compounds and the antioxidant activity as well as free radical scavenging effects of dry matter of cultural medium (DMCM), filtrate (DMF) and different solvent extracts of mycelia from Antrodia camphorata in submerged culture (ACSC). DMF showed the strongest inhibition of lipid peroxidation as a function of its concentration, and was comparable to the antioxidant activity of BHA at the same concentration of 0.2 mg/ml. The hexane extract of mycelia had the weakest antioxidant ability, whereas other mycelial extracts exhibited a modest inhibition of lipid peroxidation. DMF and water extract of mycelia (WEM) showed marked activity in free radical scavenging. The antioxidant activities of filtrate and mycelial extracts were correlated to the presence of total polyphenols, the crude triterpenoid and the protein/polysaccharide ratio of the crude polysaccharide. It was found that DMCM had lower antioxidant ability than DMF in different model systems; indicating that the major antioxidant components in DMF must be derived from the secondary metabolites of mycelia. The results presented herein indicated that DMF could possibly act as a chemopreventing agent with respect to free radical-related diseases.
Chapter 3 focuses on studies of the protective effects of DMF from Antrodia camphorata in ACSC on H2O2-induced cytotoxicity in HepG2 and carbon tetrachloride (CCl4)-induced hepatotoxicity in SD rats, and the possible mechanisms involved in this protection were also investigated. The preliminary study showed that the inhibitory effect of DMF and its crude triterpenoids on lipid peroxidation was in a dose-response manner in AAPH/linoleic acid system. HepG2 cells were pretreated with DMF at the concentration of 0.10 mg/ml for 4 h, significantly (p<0.05) decreased the lipid peroxidation which was measured by the formation of malondialdehyde induced by 1 h treatment of H2O2 (100 M). The oral pretreatment with DMF (0.25 and 0.50 mg/kg b.w.) for 5 consecutive days prior to the administration of a single dose of 40 % CCl4 (0.10 ml/100g b.w., ip) significantly prevented the increase in serum levels of hepatic enzyme markers (alanine and aspartate aminotransferase) and liver lipid peroxidation (p<0.05). The histopathological evaluation of the rat liver revealed that DMF reduced the incidence of liver lesions including neutrophil infiltration, hydropic swelling and necrosis induced by CCl4 in rats. Moreover, GSH-dependent enzymes (Glutathione peroxidase, Glutathione reductaser and Glutathione S-transferase) and the GSH/GSSG ratio were significantly improved in the oral pretreatment DMF of rats (p<0.01). Based on the above results, we speculate that DMF may play a role in preventing oxidative damage in living systems by up-regulating hepatic GSH-dependent enzymes to preserve the normal GSH/GSSH ratio and directly scavenging free radicals formed during CCl4 metabolism.
Chapter 4 focuses on the studies of the effect of methanolic extracts of mycelial (MEM) inhibited cell viability and the mechanism of MEM-induced cytotoxic in hepatoma cells. The IC50 of MEM on the proliferation of HepG2 (wild type p53) and Hep3B (delete p53) was 49.5 and 62.7 g/ml, respectively, on day 2. The effect of MEM on inhibiting cell viability was about 0.2-fold of that methanolic extracts of Antrodia camphorata fruiting bodies (MEAF). There is no observable cytotoxicity of MEM in Chang liver cells and rat primary hepatocytes at the concentration of 100 g/ml. MEM-mediated HepG2 and Hep3B cells death by apoptosis were determined by cell morphological changes, DNA fragmentation and cell cycle analysis. Cell cycle analysis revealed that MEM induced apoptosis on HepG2 via G0/G1 cell cycle arrest. HepG2 and Hep3B treated with MEM (100 g/ml) for 72 h, the apoptotic cells were 98.3 and 39.5%, respectively. It was speculated that MEM-induced cells apoptosis in HepG2 was mediated by p53-dependent that was why HepG2 was more susceptible than Hep3B. The results suggest that MEM induced HepG2 apoptosis through inhibition cell growth and up-regulation of Fas/FasL to activate the pathway of caspase-3 and -8 cascade.
Chapter 5 focuses on the studies of purification and identification of antitumor components of methanol extracts of mycelia (MEM) from ACSC. The fractions separated by HPLC from MEM were examined to evaluate the antitumor effect of inhibition cell viability on Hep3B. Then, the antitumor fraction in MEM, which was high in amount and possessed potent antitumor activity, was selected to precede purification and identification. There were six fractions (A-F) in MEM separated by analytical column of HPLC. Based on the integration of peak area, fraction C was the most abundant fraction (40%), fractions B (16%) and C (13%) was the next, and the other fraction were about 4-8% in quantity. Hep3B was treated with fraction B, C, D, and E for 24 h, and the IC50 on Hep3B was about 26-45 g/ml. However, fractions A and F showed no inhibition effect on cell viability in Hep3B. Fraction C was further separated and purified by the preparative HPLC and TLC, respectively. The molecular formula and weight of compound C were C16H22O3 and 262.2, respectively and was identified by UV, MS, IR and 1H-, and 13C-NMR.
Keywords: Antrodia camphorata in submerged culture; antioxidant; anti-tumor; apoptosis; hepatoma cells
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