|樹薯(Manihot esculenta Crantz L.)酚類化合物萃取、
Extraction and Identification of Phenolic Compounds in Cassava
(Manihot esculenta Crantz L.) and their Biological Properties
研究生：裴尤德 (Suraphan Panyod)
指導教授：邱亞伯 博士 (Albert Linton Charles)
樹薯(Manihot esculenta Crantz L.)為世界前十大熱帶糧食作物之ㄧ，其主要的澱粉成分被廣泛用於工業與食品上。樹薯製成的麵粉具有亮黃色澤，引起我們研究其活性二次代謝物作為日常食品之動機。烘乾之甜樹薯塊莖以不同比例組合之有機溶劑製備其自由態、可溶性及不可溶性酚類萃取物，並研究其酚類化合物含量及自由基清除能力。萃取物的總酚類化合物含量是依據Folin-Ciocalteu方法測定。結果顯示：乾燥和新鮮甜樹薯塊莖分別以不同比例組合的丙酮/甲醇/水（12/4/4）及丙酮/甲醇（14/6）作為萃取溶劑時，具有較高的酚醛化合物的產量和沒食子酸總酚含量(gallic acid equivalent, GAE)。乾燥與新鮮之甜樹薯塊莖自由態酚萃取物的酚類化合物含量的GAE分別為6.61-60.75和3.86-22.94 mg/g 。酚類化合物含量明顯高於分別以丙酮/甲醇/水（4/12/4）及丙酮/水（6/14）萃取的乾燥或新鮮樹薯之可溶性酚類萃取物，其可溶性酚萃取物的GAE分別為9.36-87.63和2.19-18.32 mg/g。以丙酮/甲醇/水（12/4/4）之溶劑組合可得到最高含量之不溶性酚類化合物，其GAE分別為0.45-21.07和10.39-24.12 mg/g。
將各種溶劑萃取組合中總酚類化合物含量最高的萃取物進行其色譜定量檢測及抗氧化活性試驗。高效液相色譜分析顯示，乾燥樹薯塊莖的自由態、可溶性和不溶性酚類萃取物和新鮮樹薯塊莖的可溶性和不溶性酚類萃取物的含有4 -羥基苯甲酸、兒茶素、香草酸和表兒茶素。然而，沒食子酸、槲皮素和kaempferol只有在新鮮樹薯自由態酚類萃取物中被發現。此外，主要的HPLC色譜的未知化合物經由 1H NMR和13C NMR分析證實，確定自由酚類萃取物的一個未知化合物為東莨菪素（7-hydroxy-6- methoxychromen-2-one）。
DPPH自由基清除試驗的分析結果顯示，在625 mg/mL濃度下，新鮮樹薯自由酚萃取物比其他萃取物具有較佳之自由基清除活性。在ABTS自由基抑制系統，312.5 mg/mL之新鮮樹薯萃取之自由酚和乾燥樹薯之不溶性酚類萃取物具有最高的自由基清除活性。ABTS的抑制效果隨著萃取物濃度的增加而呈現正相關，維生素 C的抑制能力高於新鮮和乾燥樹薯萃取物，新鮮樹薯自由酚和乾燥樹薯不溶性酚萃取物之抑制率與維生素E相當。Trolox的當量抗氧化能力（TEAC）系統亦顯示TEAC值與萃取物濃度呈現正相關。1250 mg/mL濃度下，新鮮樹薯自由酚萃取物較其他萃取物具有最高的抑制能力。在氧化自由基吸收能力（ORAC）系統，增加新鮮和乾燥樹薯酚類萃取物濃度會增加ORAC值。在625 mg/mL濃度下，新鮮樹薯自由酚類萃取物較其他萃取物具有最高的抑制能力。然而，萃取濃度在2500 mg/mL時，氧自由基清除能力呈現下降的趨勢。這可能是由於該方法的侷限性，高濃度值呈下降趨勢。
Cassava, Manihot esculenta Crantz L., is one of the most important tropical root crops in the developing world and its major starch component finds wide use in the industrial and food sectors. The bright yellow pigment observed in cassava-wheat noodles formed the rationale for investigating secondary metabolites that may contribute to the functional and bioactive properties of the cassava as a dietary food. The objectives of this study were to determine the best extracting solvents or their combinations in a modified extraction process, to isolate and identify the phenolic compounds, and to examine their bioactive properties.
Sweet cassava tuber root was extracted using acetone, methanol, water, and in various combinations to collect free, soluble, and insoluble-bound phenolic extracts. The contents of total phenolic compounds in the extracts were determined spectrophotometrically according to the Folin-Ciocalteu method. The results indicated the potential solvents, which had higher phenolic compound yields as gallic acid equivalent (GAE) in free phenolic extract, were solvent combinations 12:4:4 (acetone:methanol:water) and 14:6 (acetone:methanol) for dried and fresh cassava, respectively. The ranges of phenolic compound of dried and fresh sweet cassava free phenolic extracts were 6.61-60.75 and 3.86-22.94 mg GAE/g extract, respectively. Phenolic compounds were noticeably higher using 4:12:4 (acetone:methanol:water) and 6:14 (acetone:water) solvent combinations, for dried cassava and fresh cassava soluble phenolic extracts, respectively. The ranges of total phenolic compounds in soluble phenolic extract for dried and fresh sweet cassava were 9.36-87.63 and 2.19-18.32 mg GAE/g extract, respectively. Insoluble-bound phenolic compounds extracted with water were highest for dried cassava and the combination 12:4:4, (acetone:methanol:water) for fresh cassava. The ranges of the total phenolic compound in insoluble-bound phenolic extract of dried and fresh sweet cassava were 0.45-21.07 and 10.39-24.12 mg GAE/g extract, respectively.
Total phenolic compounds in dried cassava were higher than those in fresh cassava phenolic extracts, possibly due to the higher mass of dried cassava flour and drying apparently allowed the secondary metabolites to be more bio-available. Thus, the solvent extracts and the various solvent combinations that contained the highest total phenolic compounds were selected for quantitative chromatographic and antioxidant activity assays. HPLC analyses indicated that based on the phenolic standards used in the analysis, free, soluble, and insoluble-bound phenolic compounds of dried cassava phenolic extracts and soluble and insoluble phenolic extracts of fresh cassava consisted of 4-hydroxy benzoic acid, catechin, vanillic acid, and epicatechin. However, gallic acid, quercetin, and kaempferol were found only in fresh cassava free phenolic extracts. Moreover, major peaks of HPLC chromatograms indicated the presence of unknown compounds. 1H NMR and 13C NMR analysis confirmed the structure of an unknown compound in free phenolic extract as scopoletin (7-hydroxy-6-methoxychromen-2-one).
DPPH analysis indicated that the concentration 625 µg/ml of fresh cassava free phenolic extract had the highest free radical inhibition when compared to other extracts. In the ABTS free radical inhibition system, 312.5 µg/ml extract indicated the fresh cassava free phenolic and dried cassava insoluble-bound phenolic extracts had high free radical scavenging activity. ABTS inhibition increased with corresponding increases of extract concentrations of both dried and fresh cassava. Although Vitamin C demonstrated higher inhibition capacity than both fresh and dried cassava extracts, Vitamin E exhibited inhibition levels equivalent to fresh cassava free phenolic and dried cassava insoluble-bound phenolic extracts. Trolox equivalent antioxidant capacity (TEAC) analysis also indicated that increasing extract’s concentrations resulted in increased TEAC values. At the concentration of 1250 µg/ml, the fresh cassava free phenolic extract had the highest inhibition compared to other extracts. In the oxygen radical absorbance capacity (ORAC) system, increases in concentrations of fresh and dried cassava phenolic extracts tended to increase the ORAC as trolox equivalent. At the concentration of 625 µg/ml extract, fresh cassava free phenolic extract exhibited the highest inhibition compared to other extracts. However, extract concentration of more than 2500 µg/ml reveal a decrease in oxygen absorbance capacity. This was possibly due to limitation of the method to high concentrations since the value tended to decrease at similar high concentrations of trolox as standard.
The study was able to confirm the presence of phenolic compounds and make a considerable contribution to the crop’s nutritional quality of food, which will change the perception of the cassava as a dietary component.
Keywords: Manihot esculenta, cassava, phenolic compounds, antioxidant activity, ORAC