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Hot Pressurized Fluids Extraction of Functional Compounds from Taiwan Pine Needles and Barks and Associated Anti-oxidative Abilities
hot pressurized fluids extraction
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第一部分實驗，以台灣五葉松針(PM)為原料，使用L18直交表實驗設計法進行，利用酵素水解與熱壓流體(HPFE)萃取酚酸類生物活性成分。實驗結果顯示，纖維酵素、果膠酵素、半纖維素及蛋白水解酵素在連續或批式製程中皆有助於植物細胞結構組織的鬆解。酵素浸漬處理後，以連續熱壓流體萃取製程的最適當條件為使用混合酵素1.5％wt於70%乙醇水溶液（pH 6）中，在689 kPa壓力與343 K 溫度下，以10 mL/min流量萃取進行180分鐘，每克萃出物中酚酸含量可達39.80mg，而批式製程可得31.34 mg，萃提率可提升27%。五葉松(PM)與二葉松(PT)針萃出物在乙酸乙酯可溶部份，皆具有最強的抗氧化能力。PT萃出物在清除DPPH自由基、抑制過氧化自由基、清除一氧化氮自由基、螯合二價鐵離子與還原力能力等五項皆優於PM；在抑制脂質過氧化能力上，則是PM優於PT。使用化學發光法就萃出物的水溶性與油溶性部分進行抗氧化能力檢測，顯示每克PM萃出物的抗氧化能力分別相當於0.78mmol VC、1.63 mmol VE 優於PT萃出物 0.29mmol VC 、1.48 mmol VE。兩種萃出物在抑制人類癌細胞株增殖能力方面，對AGS胃腺癌與A549肺癌兩種細胞隨劑量的增加具有增生趨勢，其中AGS細胞特別顯著；對A431皮膚表皮癌細胞、DU145前列腺癌及DBTRG腦膠質母細胞瘤等三種，抑制細胞增殖能力皆隨劑量呈正相關性，在不影響對照組胚胎細胞下，分別對於人類腦膠質母細胞瘤DBTRG細胞株有10% 與 16% 的抑制增殖作用。
第二部分以台灣五葉松樹皮生物活性成分之分離與抗氧化能力評估為主；不同樹齡樹皮之甲醇萃取物，以乙酸乙酯可溶部份活性最高。化學抗氧化評估，顯示清除DPPH自由基以低樹齡樹皮（1～3年）為佳，其餘在抑制過氧化自由基釋出、抑制脂質過氧化自由基、清除一氧化氮自由基、螯合二價鐵離子與還原力等五種抗氧化效能皆隨樹齡（15～20年）成正相關性。高樹齡甲醇萃取物經正相層析分離純化後，以榭皮素（Quercetin）為，分別以化學法與化學發光法進行篩選，發現EF-18(7.13mmol VC/g、5.07 mmol VE/g)，EF-14 (0.61 mmol VC/g、9.57 mmol VE/g) 等兩份，總抗氧化能力高於榭皮素（Quercetin）(5.57 mmol VC/g、3.88 mmol VE/g)，分別達1.29、1.08倍。酚酸化合物影響總抗氧化能力之強弱，依序為結構型態、羥基取代位置有關；構型以羥肉桂酸型>黃烷醇型>羥苯甲酸型>花青素型、黃酮醇型>二羧酸型；黃烷醇型取代位置以B環上具有鄰位二羥基者為佳。
Pine species are rich in polyphenols and are important for regenerating biomass that have increasingly attracted attention in the fields of nutrition, health and medicine. In this study, two parts of experiments have been carried out. we are interested in using pine needles and pine barks of wood residue from the agricultural and forestry industries in Taiwan. Firstly, Enzymatic hydrolysis on the hot pressurized fluid extraction (HPFE) of polyphenols from the needles of Pinus taiwanensis (PT) and Pinus morrisonicola (PM) were carried out. A mixture of cellulose, hemicellulase, pectinase and protease is able to enhance plant cell breakage. Hence, these enzymatic catalyzed HPLE process dramatically reduce the extraction pressures and temperature. The results showed that the optimum conditions of two step extraction process were as follows: 1.5 % wt of mixed enzyme in 70% ethanol (pH 6) at a flow rate of 10 mL/min, under pressure 689 kPa and 343 K for 180 min. The maximum contents of polyphenols obtained was 39.8 mg per gram of extract, and the continuous hot pressurized ethanol aqueous solution was superior to batch 31.34 mg. The extracts from PT needles possessed higher antioxidant activities against DPPH radical, superoxide anion radical (O2−) and nitrite oxide radical (NO), and higher reducing ability of chelating ferrous cations (Fe2+) than those from PM needles. Particularly, the ability of scavenging NO by the PT needle extracts is more than 1.8 times better than that of scavenging by the PM needle extracts. However, the PM needle extracts inhibited peroxide radical (O22−) much better than PT needle extracts did. In addition, the total anti-oxidative capacity determined by photochemiluminescence method, the PM needles extracts demonstrated higher than that from the PT needles extracts did. In the bioassay tests, the extracts of PM and PT needles exhibited 10% and 16% of growth inhibition effect on DBTRG-glioblastoma cell of the human brain, respectively. Secondly, Expermental results showed that the ethyl acetate partition of the shoot, offshoot and trunk of PM bark of the methanol extracts was superior to the n-hexane, n-butanol and water partitions on antioxidant efficacy. The shoot extract showed excellent ability for screening DPPH free radical. But the extract of trunk showed the most obvious abilities for inhibiting lipoid peroxide, screening nitrite oxide radical and superoxide anion radical, and these abilities were all positively dependent on the growth period of tree. Furthermore, the methanol extract of trunk bark was subject to silicon chromatographic separation, and the anti-oxidative ability of the eluent was examined. The total anti-oxidative capacity of EF-14 and EF-18 fractions was superior to quercetin. Furthermore, comparing with antioxidation of the needle, the truck bark presents better anti-oxidation ability in the extracts of Pinus morrisonicola. Anti-oxidative ability is mainly related to the contents of phenols in the extracts. The bark primarily contained condensed tannins and the needle segment mainly hydroxycinnamates and flavonoids.
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