萃取方式對蛋黃果 (Pouteria campechiana) 多酚組成與其抗氧化能力之影響

Effect of Extraction Methods on Polyphenolic Constituents and Antioxidant Capacity of Eggfruit (Pouteria campechiana)

陳阮日科(Tran Nguyen Nhat Khoa)

指導教授 ： 邱亞伯 ； 林信宏

國立屏東科技大學/國際學院/熱帶農業暨國際合作系/碩士(2016年)

摘要

蛋黃果在許多熱帶地區，例如：中南美洲、東南亞國家、臺灣及澳洲，是一種商業上尚未充分利用之水果。此外，有些基因型可能被認為是不同的物種，但顯然地無此物種存在的品種分類。在臺灣，這裡有按照蛋黃果外型分類的三種生物型：1. 紡錘形、2. 圓卵形及3. 較小尺寸的圓形。蛋黃果據說被認為是一個豐富二次代謝產物之來源，尤其是酚酸與類黃酮化合物，使得其具有優異營養與抗氧化成份之潛力性功能性食品。因此，本研究的目的是研究溶劑 (丙酮、乙醇、甲醇及水) 與不同溶劑組合之的有效性，與從三種生物型之蛋黃果萃取抗氧化物質 [總酚 (TPC) 與 總類黃酮 (TFC)] 之 技術 [凍乾 (冷凍乾燥) 與 超音波]。兩種從鮮果與冷凍乾燥樣品之 高酚類含量萃取物被使用作為酚類組成份分析 (高效液相色譜-HPLC) 與生物活性評估 (總抗氧化活性、氫過氧化物清除、抗自由基、超氧陰離子清除及一氧化氮清除之檢測)。
以80%丙酮萃取新鮮蛋黃果樣品表現出最高總酚含量：21.43 mg gallic acid equivalent (GAE)/g (生物型 3)、16.48 mg GAE/g (生物型2) 及 13.69 mg GAE/g (生物型 1)。在萃取技術之篩選，以丙酮與凍乾之結合有最大之總酚含量 (31.16–46.64 mg GAE/g) 與總類黃酮含量 (4.40–4.98 mg catechin equivalent CEQ/g)。同樣地，丙酮溶液中也顯示最大之總類黃酮含量 (3.42–5.44 mg CEQ/g 乾燥物重 (DW)、4.40–4.98 mg CEQ/g 乾燥物重)。因此，藉由使用丙酮溶液之三種蛋黃果生物型的鮮果與凍乾樣品萃取物，被選擇作為進一步HPLC分析，與評估其體外抗氧化特性。
五個主要酚類物質從鮮果與凍乾樣品中被鑑定，包括：沒食子酸(3.21–4.19毫克/克 乾燥物重)、對 - 羥基苯甲酸 (0.49–3.01毫克/克 乾燥物重)、香草酸 (2.95–3.77毫克/克 乾燥物重)、丁香酸 (3.09–5.86毫克/克 乾燥物重) 及阿魏酸 (0.02–3.21毫克/克樣品 乾燥物重)。在這三種生物型之間，從生物型1的凍乾萃取樣品顯示，個別酚類化合物中，沒食子酸、香草酸及丁香酸的含量最高。再者，在液相層析四極柱式/飛行式串聯質譜儀 (LC-QToF-MS)，有些酚類化合物被鑑定出，如間苯三酚、對羥基苯甲酸、脫落酸、奎尼酸、香豆酸、莽草酸及羥基酪醇。
凍乾樣品 (100 mg/mL) 通常表現出最高抗氧化活性；例如：總抗氧化活性 (50.90–64.14 %)、過氧化氫（H2O2）清除活性 (58.39–62.23 %)、抗自由基活性 (89.78–90.15 %)、超氧陰離子自由基 (•O2-) 清除活性(89.65–97.35，在5.33 mg/mL)、一氧化氮清除活性 (51.39–70.90 %) 及氧自由基吸收能力 (ORAC) (47.14–52.18 μM TE)。
此外， 在TPC、TFC、總抗氧化及抗自由基活性，建立高相關性 (r2 ≥ 0.800)。另外，在酚與類黃酮含量顯著差異，也部分地證實三種生物型之間的差異，導致在三種蛋黃果生物型，果實之不同抗氧化特性。特別是在使用從凍乾樣品萃取樣品之抗氧化分析中，生物型1通常表現出最高的抗氧化活性，其次依序為生物型3與生物型2。結果也證實蛋黃果是具有潛力的健康食品。

關鍵字

蛋黃果 ； 酚類萃取 ； 抗氧化活性 ； 沒食子酸 ； 功能性食品

並列摘要

Eggfruit (Pouteria campechiana) is a commercially underutilized fruit in many tropical regions such as Central America, Southeast Asian countries, Taiwan, and Australia. Moreover, there are some genotypes that could be considered as different species, but apparently no varietal classification of this species exists. In Taiwan, there are three biotypes of eggfruit classified based on the shape of the fruit: spindle-shaped (1), ovoid ripe (2), and round eggfruits with smaller size (3). Eggfruit is reportedly considered as a rich source of secondary metabolites, especially in phenolic acids and flavonoids, which makes it a potential functional food with excellent nutritional and antioxidant components. Hence, the objectives of this study were to investigate the effectiveness of solvents (acetone, ethanol, methanol, and water) and a combination of different solvents, and techniques [lyophilization (freeze-drying) and ultrasonication] on the extraction of antioxidant compounds (total phenolic (TPC) and total flavonoid (TFC)] from fruits of the three biotypes. Two extracts with the highest phenolic content from fresh and freeze-dried samples were used for phenolic composition analysis (high performance liquid chromatography - HPLC) and biological activity evaluation (total antioxidant activity, hydrogen peroxide scavenging, anti-radical, superoxide anion scavenging, and nitric oxide scavenging assays).
Fresh eggfruit samples extracted with acetone 80 % exhibited the highest total phenolic contents: 21.43 mg gallic acid equivalent (GAE)/g for biotype 3; 16.48 mg GAE/g for biotype 2, and 13.69 mg GAE/g for biotype 1. In the screening of extraction techniques, the combination of acetone and lyophilization had the maximum total phenolics content (31.16–46.64 mg GAE/g) and total flavonoids content (4.40–4.98 mg catechin equivalent CEQ/g). Similarly, aqueous acetone also showed the maximum total flavonoid content [3.42–5.44 mg CEQ/g of Dried Weight (DW) and 4.40–4.98 mg CEQ/g DW]. Therefore, the extracts of fresh and lyophilized samples of the three eggfruit biotypes by using aqueous acetone were selected for further analysis using HPLC and extracts from both fresh and lyophilized fruits of the three biotypes were evaluated in vitro for their antioxidant properties.
Five major phenolic compounds were identified in fresh and freeze-dried samples, including gallic acid (3.21–4.19 mg/g DW), p-hydroxybenzoic acid (0.49–3.01 mg/g DW), vanillic acid (2.95–3.77 mg/g DW), syringic acid (3.09–5.86 mg/g DW), and ferulic acid (0.02–3.21 mg/g DW). Among the three biotypes, extracts from lyophilized sample of the biotype 1 showed the highest content of individual phenolic compounds, predominantly gallic acid, vanillic acid, and syringic acid. Furthermore, in the analysis using liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QToF-MS), some other phenolic compounds were identified, such as phloroglucinol, p-hydroxybenzoic acid, abscisic acid, quinic acid, p-coumaric acid, shikimic acid, and hydroxytyrosol.
Lyophilized samples (100 mg/mL) generally exhibited the highest antioxidant activities; for example, total antioxidant activity (50.90–64.14 %), hydrogen peroxide (H2O2) scavenging activity (58.39–62.23 %), anti-radical activity (89.78–90.15 %), superoxide anion radical (•O2-) scavenging activity (89.65–97.35 %, at 5.33 mg/mL), and nitric oxide scavenging activity (51.39–70.90 %), and oxygen radical absorbance capacity (ORAC) (47.14–52.18 µM TE).
In addition, high correlations (r2 ≥ 0.800) were established among TPC, TFC, total antioxidant, and DPPH scavenging activity. Additionally, significant differences in phenolic and flavonoid contents also partly confirmed the differences among the three biotypes, resulting in the different antioxidant properties of fruits from the three eggfruit biotypes. Particularly, among the antioxidant assays using extracts from lyophilized samples, biotype 1 and 2 generally exhibited the higher antioxidant activities than those of biotype 3. The findings confirmed eggfruit as a potential health food.

並列關鍵字

Pouteria campechiana ； phenolic extraction ； antioxidant activity ； gallic acid ； functional food

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