臺灣博碩士論文加值系統

香菇(Lentinula edodes)含有許多生理活性成份，包括麥角固醇、麥角硫因、香菇多醣、香菇嘌呤。在藥理活性研究中發現香菇萃取物具有抗發炎、降低膽固醇、抗腫瘤等活性。文獻顯示菇類中的麥角固醇經由UV光或脈衝光照射可轉化為維生素D2，而在省產食用菇中，包括香菇、杏鮑菇、金針菇、鴻喜菇中，尤以香菇所含之麥角固醇含量最高。脈衝光與連續照射紫外光方法比較，脈衝光照射具有短時間高能量之優點，但照射後造成生理活性物質之破壞，或對於抗氧化及抗發炎又會造成之影響值得進一步去探討。
本研究以香菇做為實驗材料，探討脈衝光不同的照射次數0、20、40、60次 (每次之照度能量為1.309 kJ/m2)對香菇麥角固醇轉化成維生素D2之影響及分析其他生理活性物質包括麥角硫因與總酚等，並進一步探討菇粉照射0次及照射60次之照射處理後，香菇水層及乙酸乙酯層萃取物之抗發炎及抗氧化影響。
實驗結果顯示：香菇粉在受到不同照射次數後，維生素D2濃度有顯著增加的趨勢，在照射0次時，未偵測到維生素D2，照射20次、40、60次時分別為205.65 μg/g、317.67 μg/g、362.27 μg/g。麥角固醇有減少的趨勢，但與控制組比較沒有顯著差異，含量分別為5522.6、5414.0、5260.5和5117.8 μg/g；麥角硫因有增加的趨勢，但與控制組比較沒有顯著差異，含量分別為560.0、570.1、613.6和593.3 μg/g；總酚有顯著增加的趨勢，含量分別為6.91、7.07、7.24和7.42 mg/g。
評估抗發炎性質方面，主要探討照射0次及60次的粉末萃取物對於經LPS誘導巨噬細胞株RAW264.7發炎反應之影響。香菇粉萃取後可獲得四種不同萃取物包括未照射水層(NW)、已照射水層(IW)、未照射乙酸乙酯層(NEA)以及已照射乙酸乙酯層(IEA)四組，後續將探討此四種萃取物對於經LPS誘導巨噬細胞RAW264.7發炎反應之影響。
由結果可知，IW在濃度500 μg/mL時，抑制LPS誘導巨噬細胞分泌一氧化氮(NO)的效果最好，抑制率為74.30%。抑制細胞分泌細胞激素實驗方面，在抑制腫瘤壞死因子(TNF-α)中，皆以IW效果最好，在24小時中，IW濃度為1000 μg/mL時，抑制率為13.43%；在48小時中，IW為濃度750 μg/mL時，抑制率為24.91%。在抑制細胞白介素第六因子(IL-6)實驗中，在24小時中，以NW在濃度1000 μg/mL時抑制效果最好，抑制達68.05%；在48小時中，以IW在濃度1000 μg/mL時抑制效果最好，抑制率可達到89.04%。綜合上述結果，水層萃取物對於抗發炎優於乙酸乙酯層，於是後續的實驗以水層萃取物為主。
在評估抗氧化能力的實驗，濃度為20 mg/mL時，NW與IW之還原力分別為0.99與1.03，在濃度20 mg/mL時，清除DPPH之能力分別為45.69%與51.96%。螯合亞鐵離子的實驗中，兩者則是無顯著差異。
綜合本研究可得下列結論與建議：脈衝光照射次數20次(約6.7秒)時，即可顯著的增加香菇粉維生素D2之含量。此外，脈衝光照射0、20、40、60次之香菇粉，其麥角固醇與麥角硫因之含量不會減少之外，維生素D2與總酚含量可顯著增加。
水層萃取物具有良好的抗發炎及抗氧化能力，推測可能是脈衝光照射造成菇粉水溶性物質改變，但其中的有效成分及機制尚未清楚，因此，未來可在進一步研究，使用核磁共振系統來探討IW之化學結構變化。

Lentinula edodes contains many physiologically active components, including ergrsterol, ergothioine, lentinan and eritadenine. In addition, its extracts are used to be a source for a potential anti-inflammatory, cholesterol reducing, and anti-tumor activity. According to previews studies, pulsed light or continuous UV exposure method can convert ergosterol to ergocalciferol (known as vitamin D2) and increase vitamin D formation in mushrooms. There are many mushrooms , such as Lentinula edodes, Flammulina velutipes, Pleurotus eryngii and Lyophyllum shimeji, Lentinula edodes contain high level ergosterol in Taiwan. Comparing with continuous UVB light method, pulsed light method has some advantages, such as shorter period of time and high peak energy. Therefore, it is worth to evaluate the change of physiological active substances during the irradiating.
The purposes of this study were to explore the effect under different shot (0, 20, 40, and 60 pulses, and the radiant eneygy was 0.131 J/cm2 per pulse) of pulsed light irradiation of vitamin D converted to ergosterol , to analysis the physiological activity substances, including, ergothioneine and total phenols, and to evaluate the anti-inflammatory reaction, and antioxidant properties of Lentinula edodes 921.
According to the results, powder exposed to 20, 40, and 60 shots of pulsed light, could significantly increased vitamin D2 content dose-dependently. The content of ergosterol would be decreased slightly under different irritation condition, 20, 40, and 60 shots, which were 5522.6, 5414.0, 5260.5, and 5117.8 μg/g. And the content of ergothioneine had slightly increased , which were 560.0, 570.1, 613.6, and 593.3 μg/g. Total phenols content significantly increased, contents were 6.91, 7.07, 7.24, and 7.42 mg/g.
The anti-inflammation activity evaluation, we investigated the extracts of Lentinula edodes effect on macrophage celluar-RAW264.7, induced by LPS and produce NO, TNF-a and IL-6 substances. There were four kinds of extracts used in this study, included no irritated aqueous extract (NW), irradiated aqueous extract (IW) , no ethyl acetate extract (NEA) , irritation ethyl acetate extract (IEA).
Overall the results of four extracts demonstrated the IW in the concentration of 500 μg/mL, had an excellent inhibition on macrophage to produce NO, stimulated by LPS, and the inhibition ratio was 74.3%.When the concentrations were 1000 and 750 μg/mL, IW had an excellent inhibition on LPS-induced TNF-a production at 24 and 48 hours. The inhibition were 13.43% and 24.91%.When the concentrations was 1000 μg/mL, NW had an excellent inhibition on LPS-induced IL-6 production at 24 hours.The inhibition was 68.05%. When the concentrations was 1000 μg/mL, IW had an excellent inhibition on LPS-induced IL-6 production at 48 hours. The inhibition was 89.04%. According to the results mentioned before, we found that the aqueous layer extracts exhibited better effect than ethyl acetate extracts. Therefore, aqueous layer extract were used in the following experiment.
At 20 mg/mL NW and IW, the reducing power were 0.99 and 1.03, whereas, scavenging abilities of aqueous layer extract on DPPH were 45.69% and 51.96%. On the chelating ferrous ions, there was no significant difference between NW and IW.
Overall, powder exposed 20 shots of pulsed light could significantly increase vitamin D2 content, which needed 6.7 seconds. In addition, it can also significantly enhanced the content of vitamin D2 and total phenols, and there were no influence on the content of ergosterol and ergothioneine after irritation.The anti-inflammation of irritated aqueous layer extract are better. Accoding to the previous results, we supported that the composition of aqueous layer extracts has been changed after irritation, but the mechanisms is unknown. Therefore, using NMR spectroscopy to clarify the changes of chemical structure on irritated aqueous layer extract could be proceeded in the future.

表次 viii
圖次 ix
前言 1
文獻整理 3
一、 香菇簡介 3
二、 食藥用菇之維生素 5
(一) 利用照射刺激提升食藥用菇維生素D含量 5
(二) 維生素D 6
三、 食藥用菇之生理活性物質 6
(一) 麥角硫因 6
(二) 麥角固醇 9
四、 誘導發炎反應以及其他相關因子 11
(一) 發炎反應 11
(二) 巨噬細胞 11
(三) 脂多醣 11
(四) 巨噬細胞活化之機制 12
(五) 一氧化氮 12
(六) 巨噬細胞RAW264.7受到發炎訊號後所產生的物質 16
五、 抗氧化特性 19
(一) 自由基種類 19
(二) 自由基對生物體損傷之機制 20
(三) 氧化抑制 22
(四) 抗氧化劑原理 22
(五) 天然抗氧化物質 23
研究動機 24
實驗架構 26
材料與方法 27
一、 實驗樣品 27
二、 脈衝光照射系統 27
三、 生理活性物質分析 27
(一) 維生素D2 27
(二) 麥角固醇 28
(三) 麥角硫因 29
(四) 總酚含量 30
四、抗發炎性質之評估 30
(一) 細胞株 30
(二) 試藥 30
(三) 萃取物製備與濃度 31
(四) 萃取物(IW、IEA)維生素D2之測定 31
(五) 細胞株培養 31
(六) 細胞存活率 32
(七) 一氧化氮含量之測定 33
(八) 細胞激素(TNF-α、IL-6)分析 33
五、抗氧化能力 34
(一) 還原力 34
(二) 清除1,1-二苯基-2-苦胼基團(DPPH)之能力 34
(三) 螯合亞鐵離子之能力 35
六、統計分析 35
結果與討論 36
一、 生理活性物質分析 36
(一) 維生素D2 36
(二) 麥角固醇 36
(三) 麥角硫因 38
(四) 總酚 40
二、 萃取物之抗發炎性質 42
(一) 樣品萃取率 42
(二) 萃取物(IW、IEA)之維生素D2 44
(三) 細胞存活率 44
(四) 一氧化氮含量測定 48
(五) 細胞激素分析(TNF-α) 52
(六) 細胞激素分析(IL-6) 57
三、 水層萃取物之抗氧化 75
(一) 水層萃取物還原力 75
(二) 水層萃取物之清除DPPH自由基 75
(三) 水層萃取物之螯合亞鐵離子 77
(四) 水層萃取物之抗氧化性質EC50 80
結論 84
參考文獻 86

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