超臨界流體應用於柑橘成分萃取暨毛豆異黃酮變化探討

Development of supercritical fluid extraction for nobiletin and tangeretin from Citrus species ＆ studies on the isoflavone content and distribution in vegetable soybean

10.6346/NPUST.2009.00294

李穎宏

CO2超臨界流體萃取 ； 扁實檸檬 ； 金桔 ； 乙醇 ； 川陳皮素 ； 桔皮素 ； 毛豆 ； 異黃酮 ； 播種期 ； 生長期 ； 栽培地區 ； 胚軸 ； supercritical carbon dioxide extraction ； Citrus depressa ； Citrus mitis ； ethanol ； nobiletin ； tangeretin ； vegetable soybean ； isoflavone ； sowing date ； growth period ； cultural location ； hypocotyls

屏東科技大學食品科學系所學位論文

2009年

博士

黃卓治 博士

繁體中文

論文摘要內容： 本論文分為2部份，第1部份在探討各萃取參數對超臨界流體萃取柑橘屬中Nobiletin與Tangeretin的產量影響。第2部份在探討播種期、生長期間及栽培地區對毛豆品種 (系) 及種子部位中異黃酮含量與分佈的影響。 為發展以二氧化碳超臨界流體(Supercritical carbon dioxide； SC-CO2)萃取扁實檸檬 (Citrus depressa Hataya) 與金桔 (Citrus mitis Blanco) 果皮中Nobiletin與Tangeretin。本部份探討項目包括有：共溶劑的種類、共溶劑中的水比例、共溶劑在超臨界流體的添加比例、萃取溫度、萃取壓力、CO2流速、原料粒徑大小等因子，對Nobiletin與Tangeretin萃取產量的影響。而一般傳統固液溶劑萃取法亦一併進行，以作為對照比較。萃出物的Nobiletin與Tangeretin含量以HPLC分析定量，其結構並經液相層析串聯式質譜 (LC-MS/MS) 確認。結果顯示：扁實檸檬之Nobiletin與Tangeretin最適超臨界流體萃取條件為：溫度：80℃；壓力：30 MPa；CO2流速：3.0 mL/min；原料粒徑：0.375 mm；共溶劑：85%乙醇水溶液；共溶劑添加比例：9.1%。至於，金桔之Nobiletin與Tangeretin最適SFE萃取共溶劑、共溶劑添加比例、溫度、壓力、CO2流速、原料粒徑，則分別為85% 乙醇水溶液、9.1%、80℃、 20 MPa、3.0 mL/min、 0.188 mm。相較於傳統固液溶劑萃取產量 (100%)，利用SFE自扁實檸檬、金桔萃取所得之Nobiletin與Tangeretin產量分別為108%、111%。因此，利用添加乙醇水溶液之CO2超臨界流體萃取扁實檸檬、金桔果皮中之Nobiletin與Tangeretin為兼顧環保而有效的方法。 為發展台灣毛豆產業，本部份針對播種期、生長期間及栽培地區對毛豆品種(系)及種子部位中異黃酮含量與分佈的影響進行探討。12種異黃酮以HPLC進行定量分析，為評估異黃酮分佈變化，並區分成7個群組分別為TDin、 TGlin、 TGin、 MalGly、 Gly、 AceGly及AGly。試驗結果顯示：毛豆品種(系)及種子部位中異黃酮含量，會受播種期、生長期間及栽培地區等因子影響，基因在毛豆異黃酮含量扮演著重要角色。在種子充實期 (seed filling)，全豆 (whole seed)、胚軸及子葉，在較低溫度具有含量較高之異黃酮，而較高溫度異黃酮含量較低。胚軸具有相當高的異黃酮含量，且glycitein及其另外3種衍生異黃酮，幾乎僅存在此部位內。就7個ISF群組比例大小而言，相同毛豆品種在不同播種期、不同生長期，具有不同的異黃酮分佈。然而，在胚軸及子葉中，則不受播種期與生長期影響。2個栽培地區之異黃酮群組分佈皆具有TGin ＞TDin＞TGlin的型態，但依MalGly、 Gly群組比例大小來看，則會因栽培地區的不同而不同。

The Contents of Abstract in This Thesis: The studies were composed of two parts. In part Ⅰ, the influence of several operating parameters on yield of supercritical fluid extraction (SFE) for nobiletin and tangeretin from Citrus species peel was investigated. In part Ⅱ, the change of isoflavone (ISF) content and composition in vegetable soybean varieties and their seed tissues was analyzed during two sowing date, five growth period and at two cultural location. The optimal conditions of supercritical fluid extraction for two polymethoxyflavones, nobiletin and tangeretin, from Citrus depressa Hayata and Citrus mitis Blanco were developed. The effect of modifier type, modifier composition in terms of water content in ethanol, modifier percentage, extraction condition (temperature, pressure and CO2 flow rate) and particle size on the yield of nobiletin and tangeretin were investigated. The conventional extraction method was conducted in parallel for comparison. The contents of nobiletin and tangeretin were determined by HPLC, and their molecular weights and chemical structures were also confirmed by LC-MS/MS. Results showed that: The optimal condition for selective SFE of nobiletin and tangeretin from Citrus depressa Hayata was temperature of 80℃, pressure of 30 MPa, CO2 flow rate of 3.0 mL/min and particle size of 0.375 mm with 9.1% modifier (ethanol: water = 85: 15, v/v). However, the optimal modifier composition, modifier ratio, temperature, pressure, CO2 flow rate and particle size for selective SFE of nobiletin and tangeretin from Citrus mitis Blanco was 85% aqueous ethanol, 9.1%, 80℃, 20 MPa, 3.0 mL/min and 0.188 mm, respectively. Overall, the nobiletin and tangeretin yield of SFE from Citrus depressa Hayata and Citrus mitis Blanco was 108% and 111%, respectively, compared to conventional solvent extraction method (100%). Therefore, SC-(CO2 + aqueous ethanol) extraction is considered as the most environmentally friendly extraction method of nobiletin and tangeretin from Citrus depressa Hayata and Citrus mitis Blanco peel. For developing vegetable soybean in Taiwan, the influence of sowing date, growth period and cultural location on the isoflavone (ISF) contents and distribution in vegetable soybean varieties and their seed tissues were investigated. For evaluating the change of ISF distribution, twelve ISF´s in whole seeds and seed tissues were quantified by HPLC and sorted into seven ISF groups constituted by TDin, TGlin, TGin, MalGly, Gly, AceGly and AGly. Results showed that: Total isoflavone contents of vegetable soybean varieties and their seed tissues were significantly affected by sowing date, growth period and cultural location. Genetics play a significant role in ISF contents of vegetable soybean varieties. Amount of ISF in whole seeds, hypocotyls and cotyledon was higher at lower temperature and lower at higher temperature during seed filling. Higher isoflavone contents were concentrated in hypocotyles, and glycitein and its three derivates were almost existed in these seed tissue. For the order of proportion of ISF groups, different ISF distribution was observed in the same vegetable soybean variety (whole seed), in different sowing date and growth period. However, the ISF distribution in two seed tissues was independent of sowing date and growth period. Moreover, vegetable soybean varieties grown at the two location were in a same type of ISF distribution as TGin>TDin>TGlin. On the other hand, the order of proportion of MalGly and Gly in vegetable soybean varied at various cultural locations.

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