‘巨峰’葡萄果皮中對二苯代乙烯類化合物之萃取、分離與檢測之研究

Abstract

本試驗使用‘巨峰’葡萄(Vitis vinifera × Vitis labrusca ‘Kyoho’)果皮為材料，探討萃取溶液、樣品前處理以及HPLC(高效能液相層析)分離流程，對於trans-piceid與trans-resveratrol之萃取測定的影響。 利用trans-piceid與trans-resveratrol的標準品溶液與巨峰葡萄果皮萃取液以及虎杖萃取液進行HPLC試驗，使用C18層析管柱(Thermo，Hypersil-100 C18，5μm, 250 mm× 4.6 mm)分離，紫外光偵測器設定於波長320 nm偵測，試驗結果顯示，同步分析葡萄果皮萃取液或虎杖萃取液中的trans-piceid與trans-resveratrol兩種化合物時，使用40 %氰甲烷為移動相、每分鐘0.6毫升等度淋洗管柱，偵測結果無法判讀，必須以氰甲烷與水梯度淋洗，才能將萃取液中的化合物分離。比較梯度淋洗移動相的遞變速率發現，從0至72分鐘時移動相中氰甲烷濃度由10 %增加至85 % (GS = 1.04 %/min)，流速1.0 ml/min，萃取樣品中trans-piceid與trans-resveratrol成分峰才有良好的解析度。而管柱溫度提高，可縮短所有成分峰的滯留時間，但會使理論板數降低、成分峰變寬，導致解析度下降。為了有良好的解析度以避免偵測誤差，分析時將管柱設定於30℃。 試驗結果顯示，使用0.1%鹽酸之甲醇萃取葡萄果皮得到最大量trans-piceid，75%丙酮萃取出最多trans-resveratrol，乙酸乙酯則不適合用來萃取或純化這兩種物質。單獨使用100%甲醇萃取巨峰葡萄果皮與虎杖中的trans-piceid與trans-resveratrol，經由HPLC分離、紫外光偵測儀設定於320 nm測定兩物質的含量，所得數據相對標準偏差(RSD)介於4.19~5.29 %之間。 薄層層析純化樣品試驗，以ethyl acetate：water：acetic acid = 15：5：1 (v/v/v)作為展開溶劑可將待測物質與其他化合物分離，但損失達50 %。使用Amberlite XAD-2作固相萃取純化，tran-piceid與trans-resveratrol溶解於10 %甲醇時，吸附於Amberlite XAD-2的效果最好；以80 %氰甲烷洗脫，將吸附於Amberlite XAD-2樹酯上的trans-piceid與trans-resveratrol沖提出來的效果最佳。 綜合萃取、純化以及分離偵測等各部份的試驗結果，可發展出一個快速準確的分析方法，以便於測定園產品中trans-piceid與trans-resveratrol之含量。

The objects of this research were use the skin of grape (Vitis vinifera × Vitis labrusca ‘Kyoho’) to study the influence of extract solvents, sample purification and separation program of HPLC on extraction and determination of trans-piceid and trans-resveratrol. Results showed, using methanol of 0.1% hydrochloric acid as a extract solvent got the most amount of trans-piceid, 75% acetone extracts the most trans-resveratrol, and ethyl acetate was unsuitable to be used for extracting and purifying these two compounds. Using 100% methanol to extract trans-piceid and trans-resveratrol in the grape skin without purification, via HPLC separated and detected by a UV detector ( λ=320 nm ), and obtained relatively standard deviation of data (RSD) between 4.19~5.29 %. Using ethyl acetate : water : acetic acid =15 : 5 : 1 (v/v/v) as a development solvent to carry out thin-layer chromatography could separate trans-piceid and trans-resveratrol from the extracts, but lost 50%. Proceeding solid phase extraction (SPE) by Amberlite XAD-2 to purify extracts, as trans-piceid and trans-resveratrol dissolving in 10% methanol, the adsorbent ratio in Amberlite XAD-2 was best; and using 80% acetonitrile to elute, had the best efficiency to recover trans-piceid and trans-resveratrol absorbed on the Amberlite XAD-2. The study of HPLC using a C18 column (Thermo，Hypersil-100 C18，5μm, 250 mm× 4.6 mm) to separate trans-piceid, trans-resveratrol and other compounds in extracts of ‘Kyoho’ grape skin and Polygonum cuspidatum. Results of separation detected by a UV detector ( λ=320 nm ). Results of tests showed, while analysing trans-piceid and trans-resveratrol in grapes skin or Polygonum cuspidatum extracts at the same time, using 40% acetonitrile as mobile phase set at 0.6 ml/min isocratic could not separate well, and the chromatogram could not be determined. Contrarily, using acetonitrile and water as mobile phase set at 1 ml/min gradient made better separation than isocratic. To compare divers gradient steepness, gradient elution increased acetonitrile linearly from 10% to 85% over 72 min (GS = 1.04) with a flow rate of 1 ml/min made the best resolution of trans-piceid and trans-resveratrol peaks from grapes skin or Polygonum cuspidatum extracts. As column temperature increased there was a decrease in retention time of both compounds, but the theoretical plates decreased and the peaks widened resulted in resolution decreased. In order to detect well, the column set at 30℃. To develop a quick and accurate method through these results of extraction, purification, separation and detection make analyzing trans-piceid and trans-resveratrol in horticulture crops easy.