青剛櫟之細胞培養株中多酚性成分分析方法的探討
Studies on the analytical method for polyphenols from cell culture lines of Cyclobalanopsis glauca
中文摘要
青剛櫟普遍生長在台灣800 公尺以下之低海拔及平地地區，其新鮮葉中多酚性
成分含量豐富，為了方便探討不同季節、地區間青剛櫟之遺傳變異大小和變異型 式，以利選種、育林及復育之依據、醫療資源之開發，本實驗擬以青剛櫟新鮮的 葉片、枝條、種子及其細胞培養株的萃取液，建立以高效液相層析法分析其多酚 性成分，達到簡捷、穩定、精確的分析條件。
本實驗應用80%丙酮為萃取液，萃取青剛櫟及其細胞培養株中之多酚性成分，
萃取液經濃縮後，先以二氯甲烷洗去低極性物質，再以通過C18 cartridge 之方
式濾除醣類物質，最後使用逆向層析管柱、酸性的梯度移動相條件及高感度 photodiode-array 偵測器之運用，得到有效且再現性良好的分析條件。
本次實驗結果，高效液相層析分析方法方面，r2 均在 0.999 以上，最低可偵測 濃度(LOD, limit of detection) 在 2.5 ~ 10μM 間，最低可定量濃度(LOQ, limit of quantity)在 5 ~ 12.5μM 間。從葉片中檢出(+)-catechin、(-)-epicatechin、(+)- gallocatechin、(-)-epigallocatechin、procyanidin-B-4 及 rutin；枝條及種子則檢出 (+)-catechin、(-)-epicatechin、gallic acid 及少量之(+)-gallocatechin；細胞培養株中 之多酚性成分多數可測出(+)-catechin、(+)-gallocatechin 及 gallic acid。
英文摘要
Cyclobalanopsis glauca (Thunb. ex Murray) Oerst. are distributed very commen in forests from sea level to 800m throughout Taiwan island. They are rich in the polyphenolic compounds in their leaves. To study the polyphenolic components of Cyclobalanopsis glauca in different reason or area, we developed an analytical method using high-performance liquid chromatography to separate the polyphenolic compounds in the leaves, branches, seeds and it’s cell culture lines from
Cyclobalanopsis glauca.
An aqueous acetone (80%) were added to extract the polyphenols in Cyclobalanopsis glauca and it’s cell culture lines. After condensing, the extract was delipidized with dichloromethane and pass through C18 cartridge to filter out the sugars in the
samples. Then it was chromatographed on reversed-phase column with acidic gradient mobile phase system and detected with photodiode-array. We found a well-defined, reproducible system for the separation of such polyphenols in the samples.
The results shows the value of r2 for the analysis is above 0.999. The limit of detection (LOD) is in the range of 2.5 ~ 10 μM, the limit of quantity (LOQ) is 5 ~ 12.5μM. In the samples of leaves, there are (+)-catechin, (-)-epicatechin, (+)-
gallocatechin, (-)-epigallocatechin, procyanidin-B-4 and rutin were detected. About branches and seeds, we found (+)-catechin, (-)-epicatechin and gallic acid. Most of the cell culture lines, (+)-catechin, (-)-epicatechin, (+)-gallocatechin were identified.
