固體栽培樟芝甲醇萃取物抗變異自由基功效與抗血小板凝集作用探討

Bio-active Studies of Antimutagenic Potential, Reactive oxygen species scavenging and Anti-platelet aggregation Activity on the Fruit-body Methanol Extracts from Antrodia camphorata of Solid Cultivation

魏佳瑜(Chia Yu Wei)

指導教授 ： 洪清霖 ； 蘇慶華

臺北醫學大學/公共衛生暨營養學院/公共衛生學研究所/碩士(2003年)

摘要

台灣本土的獨特菌種牛樟芝( Antrodia camphorata )是新開發出的健康食品，野生樟芝瀕臨絕種取得不易，價格極為價格昂貴，至今已列為保育類植物。民間研發出人工液態菌絲體栽培樟芝，多數學者是使用液態發酵的樟芝菌絲體作為研究材料。固體栽培是新開發出來栽培技術，運用此法培育出的樟芝，較接近於野生型態的樟芝，預期其較具有和天然樟芝相似的療效，故本研究採用固體栽培樟芝為研究材料。 目前樟芝的成分、生理活性尚在分析當中，有效成份的研究以多醣類( polysaccharides )與三帖類( triterpenoids )為研究主流，已有文獻指出樟芝多醣具有增強免疫與抗癌之效、而本研究探討樟芝特有的豐富三帖成分對抗變異影響與抑制血小板凝集的情形。 本研究採生物檢定法安姆氏沙門氏菌試驗( Ames test )進行抗氧化變異效能的探討，使用菌株Salmonella typhimurium TA102作為測試菌種，佐以自由基過氧化氫( hydrogen peroxide )、羥基自由基與致突變劑茴香素( Cumene hydroperoxide )等致變原，探測甲醇萃取的樟芝三帖，是否具有預想的自由基清除之抗變異功效。本研究結果發現，當樟芝萃取物濃度為15mg/ml、10mg/ml時，對過氧化氫、羥基自由基清除與抑制其致突變效率在與對照組相較後，結果達統計上顯著抑制效果，求得其IC50約為10mg/ml、14mg/ml、6mg/ml。 在血小板凝集試驗中，甲醇萃取樟芝的濃度為7.5μg/ml、11.5μg/ml、15μg/ml時，可抑制膠原蛋白( collagen 1μg/ml )引發的凝集現象，抑制百分比為59.13 ± 3.82、64.27 ± 7.84、96.22 ± 3.44，樟芝有效成份會抑制collagen所造成的血小板細胞內鈣離子的增加現象，而減少血小板凝集狀況，其抑制效果隨著甲醇萃取樟芝的濃度增加而提高。

關鍵字

樟芝 ； 三帖類 ； 自由基 ； 抗氧化機制 ； 抗變異 ； 抗凝血

並列摘要

Antrodia camphorata, an unique species in Taiwan growing on the wood of Cinnamomum kanehirai, is a kind of newly-developed health food. The wild type of Antrodia camphorata is on the verge of extinction and is hardly to acquire so the price is scorchingly high as well. Nowadays it has already been listed as protective species. Antrodia camphorata has been developed in the culture through deep fermentation. And the majority of scientists study liquified deep fermentable mycelia of Antrodia camphorata as their research materials. On the contrary solid culture is a newly-developed technique. Antrodia camphorata cultivates by this method approximates to wild-type ones. We can expect it is similar to natural Antrodia camphorata in therapeutic effects. Thus, we used solid-cultured Antrodia camphorata as the research material in this study. For the present the constituent and bioactivity of Antrodia camphorata is still in the analysis. And now researches in polysaccharides and triterpenoids are mainstream of in effective constituent. There have been some medical references pointing out polysaccharides have effected on strengthening human immunity and resisting cancers. This study explored the methanol extracts of Antrodia camphorata effected on anti-mutation and anti-aggregation. This study adopted Ames test, a kind of bioassay, to explore the effects against oxidative mutation. We used Salmonella typhimurium TA102 as the test strain. Besides, we used hydrogen peroxide, hydroxyl free radicals and cumene hydroperoxide as mutagens to explore whether triterpenoids extracted by methanol have expectant effects on free radical scavenging. The result of this study is as follows： The suppression ratios of the treatment of Antrodia camphorata methanol extracts at 15 mg/ml and 10 mg/ml were statistically significant with control. Our study found out the IC50 were 10 mg/ml, 14 mg/ml, 6mg/ml separately while we treated hydrogen peroxide, hydroxyl free radicals and cumene hydroperoxide as mutagens. In platelet aggregation assay, when the extract concentration of Antrodia camphorata were 7.5 μg/ml, 11.5 μg/ml, 15 μg/ml, they can inhibit agglutination induced by collagen (1μg/ml). The inhibitive percentages were 59.13±3.82%, 64.27±7.84%, 96.22±3.44% separtatately. The effective constituent of Antrodia camphorata can inhibit intracellular Ca2+ mobilization which was induced by collagen, therefore it reduced platelet agglutination and the inhibition curves were rather dose-responsive.

並列關鍵字

Antrodia champhorata ； triterpenoids ； free radical ； antioxidant ； antimutagen ； anti-platelet aggregation

參考文獻

McQueen, Michael L. Cunningham.:Rapid analysis of base-pair substitution induced by mutagenic drugs through their oxygen radical or epoxide derivatives. Mutation Research, 470, 11-18, 2000 Ames, B.

J., ESR spin trapping studies into the nature of the oxidizing species fromed in Fenton reaction : pitfalls associated with the use of 5,5-dimethyl-1-pyrroline-N-oxide in detection of the hydroxyl radical, Free Rad. Res. Commun., 18, 68, 1993 Chen, C. H. and Yang, S. W., New steroid acids from Antrodia cinnamomea, a fungal parasite of Cinnamomum micranthum, Journal of Natural Products, 58, 1655-1661, 1995 Chen, H.

W. and Chiang, H. C., Three new triterpenoids from Antrodia cinnamomea, Journal of Natural Products, 58, 365-371, 1995 Cherng, I.

W., Wu, D. P. and Chiang, H. C., Triterpenoids from Antrodia cinnamomea, Pergamon, 41, 263-267, 1996 Chiang, H.

C., Wu, D. P., Cherng, I. W. and Ueng, C. H., A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia cinnamomea, Pergamon, 39, 613-616, 1995 Clapp, N.