Ames測試法探討牛樟芝萃取物之致變異性與治癌性

The Mutagenic Effect and Potential Cancer Therapy of Antrodia Cinnamomea by Ames Test

湯凱鈴(KAI-LING TANG)

指導教授 ： 王順成

朝陽科技大學/理工學院/環境工程與管理系/碩士(2016年)

摘要

牛樟芝萃取物的生物活性範圍相當廣泛，從血管舒張到抑制癌症等。其富含許多治療癌症和其他調節免疫相關疾病之化合物，如醣類 (Polysaccharides)之外、三萜類 (Triterpene)、苯類 (Benzenes)等。牛樟芝萃取成分除治療效果之外，是否尚具毒性，為許多消費者關心之議題。因此人體食用之安全性問題乃受到重視，此為本研究重點之一。本研究使用四種牛樟芝萃取物，分別為1. 液態培養樟芝菌絲體2. 洋菜培養皿樟芝菌絲體3. 牛樟椴木培養樟芝菌絲體4. 牛樟椴木培養樟芝子實體，其培養天數為14日、1個月、6個月、3至5年。四種培養型態之牛樟芝萃取物以索氏萃取進行純化，並利用HPLC分析其三萜類含量，其含量依序為牛樟椴木培養樟芝子實體＞牛樟椴木培養樟芝菌絲體＞洋菜培養皿樟芝菌絲體＞液態培養樟芝菌絲體，本實驗以Ames test利用鼠傷寒沙門氏菌之TA98和TA100菌株針對四種類型牛樟芝(Antrodia cinnamomea)萃取物進行安全性之致突變試驗測試，分別以代謝組(添加S9)與非代謝組(無添加S9)進行之。結果顯示該四種類型牛樟芝萃取物於試驗範圍(1- 1000 µg/plate)均不具致突變性，推估劑量試驗範圍內對生物體具安全性。
本研究亦同時進行功能性抗致突變試驗，結果顯示，四種培養型式之牛樟芝於代謝組(添加S9)抗突變試驗均較非代謝組(無添加S9)抑制效果佳，顯示牛樟芝萃取物經由代謝後更能發揮其抗突變效果，且隨著試驗劑量增高(1-1000 µg/plate)具有較好之抗突變效果，呈現劑量反應關係。針對鼠傷寒沙門氏菌框構位變異(Frame-shift mutation)菌種TA98之抗突變試驗中發現，三萜類含量最高之牛樟椴木培養樟芝子實體代謝組(添加S9)試驗中，低劑量(10-100 µg/plate)抑制效果與高劑量組(1000 µg/plate)效果差異不大，由於牛樟椴木培養樟芝子實體培養時間較常，所以10 µg/plate做為製劑開發之劑量較1000 µg/plate更具經濟效益。其中特別於鹽基置換變異 (Base-pair substitution) 菌種TA100對4-nitroquinoline N-oxide(4NQO)之抗突變試驗發現，具較高含量之化合物A(4,7-Dimethoxy-5-methyl-1,3-ben-zodioxole)之洋菜培養皿樟芝菌絲體萃取物抗突變試驗中，較三萜類含量較多之牛樟椴木培養樟芝子實體萃取物更具療效，且於低劑量組(1 µg/plate)相較於其他培養方式之抗突變效果高達32%，說明洋菜培養皿樟芝菌絲體萃取物比牛樟椴木培養樟芝子實體萃取物更具經濟價值。本實驗結果顯示四種不同培養型態之牛樟芝萃取物於試驗劑量範圍內(1- 1000 µg/plate)不具致突變反應且於代謝組(添加S9)具良好之抗突變效果，而生物體具代謝之功能，依實驗結果推估如人體食用代謝後具有良好之抑制癌變效果，故牛樟芝萃取物具藥物製劑開發之潛力。

關鍵字

液態培養樟芝菌絲體 ； 洋菜培養皿樟芝菌絲體 ； 牛樟椴木培養樟芝菌絲體 ； 牛樟椴木培養樟芝子實體 ； 致突變試驗 ； 抗突變試驗

並列摘要

The crude extracts of Antrodia cinnamomea. have wide biological activities, from the suppression of cancer to the diastole of blood vessel. It contains many chemicals, such as polysaccharides, triterpenes, benzenes, lignans, bezoquinones, etc., that effect against cancer and other immune diseases. However, does the extract of Antrodia cinnamomea have harmful effect to human body, in addition to its therapy effects, becomes another important concern to consumers. This research aimed on this concern and studied the triterpene contents in and the mutagenicity of the crude extract of Antrodia cinnamomea. The cancer therapy effect of the crude extract of Antrodia cinnamomea was also studied.
All four types of artificial culture of Antrodia cinnamomea including 1. liquid culture mycelium, 2. agar culture mycelia, 3. basswood cultivation Antrodia camphorata mycelia, and 4. Antrodia camphorata fruiting body basswood that had cultured for fourteen days, one month, six months, and three to five years, respectively, were tested. Crude extracts of these four types of culture were prepared by soxhlet extraction. The triterpene contents were analyzed with HPLC, and was highest in the extract of Antrodia camphorata fruiting body basswood, followed by basswood cultivation Antrodia camphorata mycelia, agar culture mycelia, and liquid culture mycelium in decreasing order.
The mutagenicity of the crude extract of these four types of culture of Antrodia were examined with Ames test using strains TA98 and TA100 of Salmonella typhimurium. The effect of liver in activating enzyme (S9) was also tested. No mutagenicity was observed in the tested range of concentration. We postulate these extracts are safe to living organisms.
The anti-mutation effects of these crude extracts were also observed to evaluate their therapy effect on cancer. Extract of all four types of the culture of Antrodia gave better antimutagentic effect when added li-ver activating enzyme (S9) than without S9. This effect showed a dose- response relationship. The anti-mutation effect of the extract of Antrodi-a camphorata fruiting body of basswood culture to the TA98 strain of Salmonella typhimurium, with the addition of S9, was similar between lower dose (10-100 µg/plate) and the high dose (1000 µg/plate). A m-edicine prepared from a low dose of 10 µg/plate would be much moreeconomic than a one from a high dose of 1000 µg/plate as Antrodia camphorata fruiting body basswood culture took much longer culturing t-ime. An inspiring result is the extract of agar culture mycelia which co-ntained more 4,7-dimethoxy-5-methyl-1,3-benzodioxole gave better antim-utagentic effe-ct than the extract of Antrodia camphor-ata fru-iting bodybasswood cul-ture which contained more triterpenes. Even one microgra-me per milliliter could gave 32% suppression on the reverse mutation of TA98, obviously higher than the 11-16% suppression of extract of ot-her types.
The results of this study showed the crude extract of all four culturing types of Antrodia had no mutagenicity in the tested range of dose, 1-1000 µg/plate, and had good antimutagentic effect when liver activating enzyme were added. Based on these results, the extract of Antrodia would be likely to provide good anti-mutation effect in human body. Therefore, it is highly potential to formulate effective medicine from the extract of Antrodia cinnamomea.

並列關鍵字

Liquid culture mycelium of Antrodia camphorata ； Agar culture mycelia of Antrodia camphorata ； Basswood cultivation Antrodia camphorata mycelia ； Antrodia camphorata fruiting body basswood culture ； Mutagenicity test ； Anti-mutation test

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