Effect of Antrodia camphorata fermentation broth cultivated for various time periods on the viabilities of tumor cells

本研究的目的為探討不同培養時間的樟芝醱酵液對六種腫瘤細胞存活率之影響，以及探討醱酵液中化合物與抗腫瘤功能之關係。 第一部分研究結果顯示醱酵二週後醱酵時間並不影響醱酵液pH 值。菌絲體含量隨著醱酵時間增加而增加，在六週時達到最高點，醱酵濾液固形物(FB)含量則隨著醱酵時間增加而減少。再者，菌絲體乙醇萃取物固形物(EEM)含量隨著醱酵時間增加而增加，在第六週時達到最高點，然後則減少；但是醱酵濾液乙醇萃取物固形物(EEF)含量則隨著醱酵時間增加而減少。菌絲體粗三萜與醱酵液粗三萜含量皆隨著醱酵時間增加而增加。然而，在第一部分研究中雖然發現所有的萃取物對於六種腫瘤細胞的生長均有抑制效果，卻未能找出樣品之醱酵時間與抑制腫瘤細胞功能之間的相關性。 第二部分研究即將樟芝之醱酵濾液及菌絲體中粗三萜萃取出來，分別探討其對腫瘤細胞生長之抑制效果。結果顯示菌絲體粗三萜(TM)對於六種腫瘤細胞生長的抑制效果隨著醱酵時間的增加而增加，但醱酵濾液粗三萜(TF) 對於六種腫瘤細胞生長的抑制效果則不與醱酵時間相關。在本實驗中找出最有效之粗三萜是自培養至第八週之菌絲體萃出之三萜(8 TM)，以及最敏感的癌細胞株為HeLa 。 本研究第三部分實驗乃將醱酵八週的菌絲體利用不同溶劑分離粗三萜分離過程中產出七個區分物，分別為Fc 1 、Fc 2 、Fc 3 、Fc 4 、Fc 5 、Fc 6 及Fc 7 ，其中Fc 1 、Fc 2 、Fc 3 、Fc 4 及Fc 7對於HeLa 細胞的生長均具有抑制效果，並且以Fc7 (即粗三萜)最為有效，其IC50 僅17.52μg/ml 。經由HPLC 分析比對推測菌絲體中所含有之二種成分，分別是Rt 33.54 及Rt 36.20 ，可能是具抗腫瘤活性的相關成分，可以作為樟芝發酵時之指標成分。The objectives of this research were to study the effects of Antrodia camphorata fermentation broth cultivated for various time periods on the viabilities of six tumor cells, and the compounds in the fermentation broth responsible for the antitumor functions. This research was carried out in three parts. In the first part of this research, the results showed that cultivation time didn’t affect the pH of the fermentation broth. The mycelium content of the fermentation broth increased with the cultivation time, but reached its maximum in 6 weeks. The solid content of the filtrate of the fermentation broth, on the other hand, decreased with cultivation time. In addition, the solid content of EtOH extract of mycelium (EEM) increased with cultivation time up to 6 weeks, and then decreased; but the solid content of EtOH extracts of filtrate (EEF) decreased with cultivation time. Moreover, the results indicated that the crude triterpenoids in both EEM and EEF increased with cultivation time. All of the extracts inhibited the growth of the six tumor cells investigated in this study, but it was unable to find out the most representative relationship between inhibition of the growth of six tumor cells by all the samples and the cultivation time in this part of the research. In the second part, the results showed that the effect of inhibition of the growth of six tumor cells by the crude triterpenes of EEM increased with the cultivation time, but the inhibitory effect by the crude triterpenes of EEF didn’t related to the cultivation time. The crude triterpenes extracted from the mycelium cultivated for 8 weeks had the most significant inhibitory effect on the HeLa cells. In the third part of this research, the mycelium cultivated for 8 weeks was extracted by different solvents to yield 7 fractions, and they were named Fc1, Fc2, Fc3, Fc4, Fc5, Fc6 and Fc7. Among these fractions, Fc1, Fc2, Fc3, Fc4 and Fc7 showed inhibitory effect on the growth of HeLa tumor cell, and Fc7 (Ps. Fc7 was also crude triterpenes.) has the lowest IC50 (17.5 μg/ml). The HPLC analysis indicated that there were two compounds (Rt 33.54 and Rt 36.24 min) existed in the mycelium of A. camphorata were highly correlated to the anti-tumor activity of the fermentation process. These two compounds might be used as marker during fermentation process.中文摘要 …………………………………………………………………………… i 英文摘要 ………………………………………………………………………… iii 前言 ………………………………………………………………………………… v 壹、文獻整理 ……………………………………………………………………… 1 (一) 癌症 ……………………………………………………………………… 1 (二) 樟芝 ……………………………………………………………………… 3 (三) 三萜類化合物 …………………………………………………………… 5 貳、實驗架構 ……………………………………………………………………… 11 叁、材料與方法 ……………………………………………………………….…… 13 一、實驗材料 ………………………………………………………………… 13 (1) 實驗菌株 ……………………………………………………………… 13 (2) 實驗細胞株 …………………………………………………………… 13 (3) 藥品 …………………………………………………………………… 13 (4) 器材與儀器…………………………………………………………….. 15 二、實驗方法 ………………………………………………………………… 17 (1) 樟芝菌株實驗 …………………………………………………………. 17 I. 培養皿平板培養 ………………………………………………. 17 II. 三角瓶液態培養菌酉元 …………………………………………… 17 III. 三角瓶液態醱酵 …...……………………………………………… 18 IV. 菌絲體乾燥與分離 ...……………………………………………… 18 V. 醱酵濾液之製備 ...……………………………………………… 19 VI. 菌絲體乙醇萃取物之製備 ……………………………………… 19 VII. 醱酵濾液乙醇萃取物之製備 …………………………………… 19 (2) 癌細胞株實驗 ...………………………………………………………… 19 I. 細胞株培養 ………………………………………………………. 19 II. 貼附性細胞之繼代培養 ……………………………………… 20 III. 半貼附半懸浮性細胞之繼代培養 ……………………………… 20 IV. 細胞株之冷凍保存 ……………………………………………… 21 V. 細胞株之解凍活化 ………………………………………………. 21 VI. 細胞計數 …………………………………………………………... 22 VII. 細胞存活率試驗( MTT assay ) ……………………………………. 22 1. 貼附性細胞之細胞存活率試驗 ………………………………… 22 2. 半貼附半懸浮性細胞之細胞存活率試驗 ……………………… 23 (3) 分析方法 ………………………………………………………………... 24 I. pH 值檢測 ………………………………………………………… 24 II. 還原醣測定 ……………………………………………………… 24 III. 多醣類含量分析 …………………………………………………. 24 IV. 粗三萜分離 ………………………………………………………. 25 V. High-performance liquid chromatograph 分析 ………………….. 26 肆、結果與討論 …………………………………………………………………… 28 第一部分 ………………………………………………………………………… 28 (一) 培養時間對樟芝醱酵液各種性質之影響 ……………………………… 28 (1) 培養時間對樟芝醱酵濾液pH 值之影響 ……………………………… 28 (2) 培養時間對樟芝醱酵液菌絲體含量之影響 …………………………… 30 (3) 培養時間對樟芝醱酵濾液固形物含量之影響 ………………………… 32 (4) 培養時間對樟芝醱酵液中菌絲體乙醇萃取物(EEM)固形物含量之影響………………………………………………………………………… 33 (5) 培養時間對樟芝醱酵濾液乙醇萃取物(EEF)固形物含量之影響……… 36 (6) 培養時間對樟芝醱酵濾液總多醣含量之影響 ………………………… 38 (7) 培養時間對樟芝醱酵濾液還原醣含量之影響 ………………………… 40 (8) 培養時間對樟芝醱酵液中粗三萜含量之影響 ………………………… 42 (二) 樟芝醱酵濾液(FB)、醱酵濾液乙醇萃取物(EEF)及菌絲體乙醇萃取物(EEM)對各種腫瘤細胞抑制之探討 …………………………………… 44 (1) 樟芝醱酵濾液、醱酵濾液乙醇萃取物及菌絲體乙醇萃取物對胃癌細胞株(AGS)抑制之探討 ……………………………………………………… 44 (2) 樟芝醱酵濾液、醱酵濾液乙醇萃取物及菌絲體乙醇萃取物對結腸腺癌細胞株(COLO 320 HSR)抑制之探討 …………………………………… 49 (3) 樟芝醱酵濾液、醱酵濾液乙醇萃取物及菌絲體乙醇萃取物對子宮頸上皮癌細胞株(HeLa)抑制之探討 …………………………………………… 54 (4) 樟芝醱酵濾液、醱酵濾液乙醇萃取物及菌絲體乙醇萃取物對肝癌細胞株(Hep3B)抑制之探討 …………………………………………………… 59 (5) 樟芝醱酵濾液、醱酵濾液乙醇萃取物及菌絲體乙醇萃取物對攝護腺癌細胞株(LNCaP. FGC)抑制之探討 ………………………………………… 64 (6) 樟芝醱酵濾液、醱酵濾液乙醇萃取物及菌絲體乙醇萃取物對乳癌細胞株(T-47D)抑制之探討 …………………………………………………… 69 第二部分 ………………………………………………………………………… 79 (一) 樟芝醱酵濾液粗三萜及樟芝菌絲體粗三萜對六種癌細胞生長之抑制現象 (1) 樟芝醱酵濾液粗三萜及樟芝菌絲體粗三萜對胃癌細胞株(AGS)抑制之探討 ………………………………………………………………………… 79 (2) 樟芝醱酵濾液粗三萜及樟芝菌絲體粗三萜對結腸腺癌細胞株(COLO 320 HSR)抑制之探討 ……………………………………………………… 83 (3) 樟芝醱酵濾液粗三萜及樟芝菌絲體粗三萜對子宮頸上皮癌細胞株(HeLa)抑制之探討 …………………………………………………………… 87 (4) 樟芝醱酵濾液粗三萜及樟芝菌絲體粗三萜對肝癌細胞株(Hep3B)抑制之探討 ……………………………………………………………………… 91 (5) 樟芝醱酵濾液粗三萜及樟芝菌絲體粗三萜對攝護腺癌細胞株(LNCaP. FGC)抑制之探討………………………………………………………… 95 (6) 樟芝醱酵液粗三萜及樟芝菌絲體粗三萜對乳癌細胞株(T-47D)抑制之探討 ………………………………………………………………………… 99 第三部分 ………………………………………………………………………… 106 (一) 樟芝菌絲體七種區分物對子宮頸上皮癌細胞株(HeLa)抑制之探討… 106 (二) 利用high-performance liquid chromatograph 分析樟芝醱酵液之成分… 109 (1) 分析條件之探討 ……………………………………………………… 109 (2) 樟芝醱酵濾液(FB) 之high-performance liquid chromatograph 分析… 113 (3) 樟芝醱酵濾液乙醇萃取物(EEF) 之high-performance liquid chromatograph 分析 …………………………………………………… 117 (4) 樟芝菌絲體乙醇萃取物(EEM)之high-performance liquid chromatograph 分析……………………………………………………………………… 121 (5) 樟芝菌絲體粗三萜之high-performance liquid chromatograph 分析… 125 (6) 樟芝醱酵液粗三萜之high-performance liquid chromatograph 分析… 120 (7) 醱酵八週之樟芝菌絲體七種區分物之HPLC 分析實驗……………… 132 伍、結論…………………………………………………………………………… 135 陸、參考文獻………………………………………………………………………137 附錄…………………………………………………………………………………14