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標題: 香杉芝分子親緣鑑定、代謝物分析及其活性探索
Phylogenetic Analysis, Metabolites Profiling, and Bioactivities Investigation of Antrodia salmonea
作者: 陳潔音
Chieh-Yin Chen
關鍵字: 香杉芝;牛樟芝;親緣鑑定;代謝物分析;生物活性;口服急毒性;Antrodia salmonea (Taiwanofungus salmoneus);Antrodia cinnamomea (Atrodia cinnamomea);phylogenetics;metabolites profiling;bioactivities;acute oral toxicity
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香杉芝 (Antrodia salmonea, syn. Taiwanofungus salmoneus; 本研究簡稱為 AS) 為臺灣特有多孔菌科 (Polyporacea) 真菌,子實體外觀與顏色均非常近似同屬之牛樟芝 (Antrodia cinnamomea, syn. Antrodia camphorata or Taiwanofungus camphoratus; 本研究簡稱為 AC),但兩者寄主完全不同。牛樟芝已知具有多種生物活性而在市場上有高價值,由於難以分辨兩者而衍生交易糾紛,且關於香杉芝之科學文獻尚開始起步,故探討其與牛樟芝之差異格外重要。本研究分別針對香杉芝與牛樟芝進行核醣體核酸序列片段的釣取與定序,確定其內轉錄間隔區序列,並利用不同軟體進行親緣關係之分析,成功利用該內轉錄間隔區序列分辨此二菌種,而該序列未來可發展成專一性的分子標誌,用以兩者之鑑識。代謝體方面,於揮發物組成分析結果顯示香杉芝子實體之揮發性成分主要為α-cedrene、1-octen-3-ol、D-limonene、cadinadiene、germacrene D、isolongifolene 及 α-muurolene;牛樟芝主要揮發性主要成分則為3,4,5-trimethoxy benzaldehyde、δ-guaiene、isolongifolene、1-octen-3-ol、4-terpinenol、α- guaiene 及 p-cymene。非揮發性成分方面可以用 5 種麥角甾烷類 (ergostane type) 及 2 種羊毛甾烷類 (lanostane) 之三萜類作為區分香杉芝與牛樟芝之指標成分。由指紋圖譜分析結果可知,香杉芝子實體含量最多的三萜為 (R,S)-antcin C (924.23 ± 4.82 μg/mg),牛樟芝子實體則為 (R,S)-Antcin B (375.91 ± 91 μg/mg)。根據本研究發現,以指紋圖譜可以鑑別香杉芝與牛樟芝,尤其是 antcin M 及 methyl antcinate K此 2 個化合物,僅出現於香杉芝的指紋圖譜中。在活性方面,香杉芝之抗發炎活性優於牛樟芝,牛樟芝則有較強之細胞毒性。最後,急毒性試驗顯示,香杉芝乙醇萃取物即使餵食高達 3 g/kg 之體重劑量連續 28 天,仍無口服急毒性症狀,顯示香杉芝乙醇萃取物之安全性。現今香杉芝常被替代牛樟芝使用,然而兩者之成分組成與生物活性均大不相同,為香杉芝取代性之安全考量,建議評估香杉芝之毒理學及生物活性,作更進一步探討。

Antrodia salmonea (AS, syn. Taiwanofungus salmoneus), is an endemic brown-rot Polyporaceae fungus in Taiwan. The appearance and color of its fruiting bodies are similar to A. cinnamomea (AC, syn. A. camphorata or Taiwanofungus camphoratus), which is also a Polyporaceae fungus; however, the habitats of AS and AC are significant different. AC is known for its various good biological activities and high value. Because of the endless trade disputesnd and the lack of studies about AS, it is very important to identify the difference of the 2 species. In this study, the ribosome DNA (rDNA) was isolated from them. It was successful used the internal transcribed spacer (ITS) sequence located in rDNA to identified AS and AC in this study. Not only the sequences were difference, the phylogenetic analysis confirmed the different between AS and AC, and ITS sequence could be a good molecular marker. We also compared the morphological observations, metabolites, and bioactivity of AS and AC fruiting bodies. The volatiles of AS and AC were characterized and α-cedrene was found to be the most abundant compound in AS; the other abundant compounds were 1-octen-3-ol, D-limonene, cadinadiene, germacrene D, isolongifolene, and α-muurolene. In AC, the main volatiles were 3,4,5-trimethoxybenzaldehyde, δ-guaiene, isolongifolene, 1-octen-3-ol, 4-terpinenol, α- guaiene, and p-cymene. Furthermore, five ergostane-type triterpenoids and two lanostane-type triterpenoids were selected as index compounds characterizing AS and AC metabolites. The content of each compound varied between AS and AC. (R,S)-antcin C (184.85±0.96 μg/mg) was the major triterpenoid in the AS fruiting body. However, (R,S)-antcin B was the most abundant compound in AC fruiting bodies (75.18±0.11 μg/mg). According to this study, it could distinguish between AS and AC by fingerprints, especially since two compounds, antcin M and methyl antcinate K, were only present in the AS fingerprint. Finally, examination of anti-inflammation activity and cytotoxicity showed that AS possessed more anti-inflammatory activity than AC; however, AS was more cytotoxic than AC. Dosage at 3 g/kg body weight, showed no acute oral toxicity by acute oral test of mice. It demonstrated the safety of EtOH extracts from AS. In conclusion, the composition and bioactivity of the fruiting bodies of AC and AS varies, therefore, it is recommended that further toxicological evaluation and investigation of the biological activity of AS is carried out to ensure its safe and efficacious use as an alternative to AC.
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