Effect of extract from solid state Grifola frondosa fermented soybean and irradiated by pulsed light on 3T3-L1 preadipocytes

Recently, people improve health awareness, obesity has become a public health issue of concern is the potential chronic disease risk factors. This shows that obesity has serious threat to human health. Maitake (Grifola frondosa ) is a popular study material of carpophore and hyphas, that has contains various bioactive substances and nutrients. Many studies have shown that Maitake mushroom exhibits ability of anti-cancer, and there are also studies that can improve for diabetes hypertension, high cholesterol, etc. In this study, first the use of pulsed light irradiation for 30 times Maitake mushroom soy solid-state fermentation products to freeze drying and milling, and then extracted with 70 % ethanol. Second, after removeing the alchol (remaining aqueous layer), extracted with ethyl acetate. It can be obtained the extacted of ethyl acetate layer (NEA: non-irradiated; IEA: irradiated) and the aqueous layer (NWA: non-irradiated; IWA: irradiated). The extract was adjusted to an appropriate range of different concentrations, and cultured in 3T3-L1 adipose. Assessment the impact of the growth extract on adipocyte differentiation before, during and after, then to determine whether 3T3-L1 adipose can inhibit the generation of adipose. The results of MTT shows (1)The ethyl acetate layer (IEA) was found, whether 24 or 48 hours culture, there are reduced fat cells viability by 48 hours is the best cell culture, cell viability was only 84.31% ;(2) In adipocyte differentiation, the different extracts (IWA and IEA) having theability to inhibitlipid content, particularly IEAextracts, at high concentration of 1000 μg/ mLthe cell viabilitywas 43.69 %；(3) The mature fatty cells co-cultured with extracts 24 hours : the waterlayer extracts (IWA), whenconcentrationincreasing, the cell viabilitydecreased slightly. At high concentration, the cell viability was 81.68 %. On Oil red O stained material (OROSM) show the 3T3-L1 adipocytes differentiate into mature cells, and then put extract (IWA and IEA). At high concentrations, the remaining survival 81.68 % and 86.11 %, respectively. In the analysis of cell cycle, the discovery 3T3-L1 preadipocytes and aqueous layer extract (IWA) co-cultured cells after 24 and 48 hours, it can inhibit the growth of fat cells situation. To give the extact of mature cells (IWA and IEA), both make the fat cells arrested on G0/G1 phase、reduce DNA replication in S phase , and G2/M phase was shut down. Thus affect the phenolic compounds ofaqueous layer can inhibit 3T3-L1 adipose precursor cell proliferation and lipogenous. The active substance in the ethyl acetate layer having the ability to inhibit the growth of mature adipocytes lipogenous. Based on the above results, this study found significantly inhibited that the aqueous layer of Grifola frondosa soy extract effect 3T3-L1 preadipocytes proliferation than the ethyl acetate layer. Expectations of future experiments, maybe can find out the exact impact of the inhibitory activity of substances 3T3-L1 adipocytes.近年來大家健康意識的提高，肥胖已成為大家關心的公共健康課題，也是慢性疾病中的潛在危險因子，這說明肥胖對人類健康存在嚴重的威脅。舞菇在這幾年成為一種熱們研究的材料，其子實體和菌絲體皆具有良好的健康食療效果，也有研究指出，對於糖尿病、高血壓、高血脂等亦有改善效果。 在本實驗中，是利用脈衝照射30下的舞菇黃豆固態發酵產物之凍乾粉末，再以70 %乙醇萃取，移除酒精後 (剩下水層)，再以乙酸乙酯萃取，可獲得水層和乙酸乙酯層萃取物。調整萃取物至適當且不同濃度後，培養於3T3-L1脂肪細胞，評估萃取物對脂肪細胞在分化前、中、後生長之影響，並評估是否可延緩細胞內油滴生成。 MTT結果顯示，(1)發現在乙酸乙酯層(IEA)中，不論24或48小時培養，皆有降低脂肪細胞的存活率，尤其是培養細胞48小時效果最好，細胞存活率只剩下84.31 %；(2)在脂肪細胞分化過程中，不同的萃取物(IWA及IEA)皆有抑制脂肪細胞脂質生成的能力，尤其是IEA萃取物，具有明顯抑制存活率效果，高濃度1000 μg/mL 下，其細胞存活率為43.69 %。 (3)而成熟脂肪細胞後與萃取物共同培養24小時，經脈衝光照射之水層樣品(IWA)，是唯一隨著濃度的增加，細胞存活率略有下降，在高濃度下，其存活率為81.68 %。 在油紅試驗(Oil Red O Stain)結果發現，將3T3-L1脂肪細胞分化為成熟細胞，再投入萃取物(IWA及IEA)，在高濃度下，分別剩下81.68 % 及86.11%的相對脂質生成量。 在細胞週期的分析中，發現3T3-L1前驅脂肪細胞與水層萃取物(IWA)共同培養細胞24及48小時後，具有抑制脂肪細胞生長情形，而造成此效果的原因為水層萃取物中所含的酚類活性化合物； (3)成熟細胞投予萃取物(IWA及IEA)皆有讓脂肪細胞G0/G1期停滯，S期之DNA複製降低，G2/M停擺的狀況。 綜合以上結果，本實驗發現舞菇黃豆水層萃取物對於抑制3T3-L1前驅脂肪細胞增生之效果明顯於乙酸乙酯層。可在後續的實驗中，進一步再找出影響抑制3T3-L1脂肪細胞的活性物質成分。目錄 前言 1 文獻整理 3 一、舞菇 3 (一)簡介 3 (二)藥用活性 3 二、脈衝光照射 4 (一)脈衝光應用 4 (二)照射與維生素D 4 三、肥胖 5 (一)肥胖的定義與成因 5 (二)肥胖的健康風險與危害 7 四、脂肪組織與脂肪細胞 7 (一)脂肪組織 7 (二)前驅脂肪細胞 9 (三)3T3-L1前驅脂肪細胞與分化相關機制 11 五、細胞週期 11 六、天然化合物對於脂肪細胞之研究 14 研究動機 16 實驗架構 17 材料與方法 18 一、實驗材料 18 二、實驗方法 19 (一)樣品製備 19 (二)3T3-L1細胞株培養 20 (三) 樣品對3T3-L1前脂肪細胞之毒性測定 21 (四)細胞週期 (Cell cycle) 22 (五)油紅染色 ( Oil red O Staining ) 22 (六)統計分析 23 結果與討論 24 一、樣品萃取率 24 二、3T3-L1脂肪細胞生長分化情形 24 三、細胞存活率試驗 28 四、油紅染色 51 五、細胞週期 62 結論 83 參考文獻 8