Transport and absorption of proanthocyanidin A2 and epicatechin from longan flower in Caco-2 cell monolayer model and the anti-inflammatory as well as antioxidant effects

許多研究發現多酚類物質的攝取能增加身體內抵抗氧化壓力的能力，而在眾多的多酚類物質中，原花青素及兒茶素被認為是較有效的自由基清除劑。本實驗室之前的研究發現，龍眼 (Dimocarpus longan Lour.) 花水萃出物及甲醇萃出物皆具有良好的抗氧化效果，其主要的活性物質為proanthocyanidin A2 (PA2) 及(-)-epicatechin (EC)。然而目前對於PA2之生物可利用率的研究仍十分有限。研究利用Caco-2細胞單層膜之模式系統探討PA2及EC在模擬人類腸道系統之吸收情形，藉以評估其生物可利用率。實驗中發現PA2相當不穩定，PA2經細胞實驗後，以HPLC分析發現會產生3個不同滯留時間的波峰，以MS及NMR鑑定出除PA2外，另兩個波峰為PA2的同分異構物epicatechin-(4β→8;2β→O→7)-ent-catechin，(PA4)；epicatechin- (4β→6;2β→O→7)- ent-catechin，(PA5)。此外由於龍眼花中的PA2及EC並非單獨存在，故本實驗以不同莫爾比或重量比的PA2:EC (2:1)、(1:1)、(1:2) 和PA2及EC在龍眼花中天然存在的比例 (1:3.3)，4個混合組；以及PA2、EC、PA4及PA5，4個純物質組，進一步探討PA2及EC以及由PA2產生的同分異構物的生物可利用率、抗氧化及抗發炎功效。aco-2細胞單層膜通透試驗結果發現PA2能被腸黏膜細胞所吸收進入trans-well下層，且隨著時間的增加，通透至下層的PA2量也就越趨增加；然而EC則完全不能通過細胞單層膜，這是由於Caco-2細胞上有對EC高專一性的外吐蛋白所致。另外，PA4 通透率 (Papp = 11.45 ± 5.02 ×10-6 cm/s) 遠高於PA2 (Papp = 4.72 ± 0.42×10-6 cm/s) 為其2.4倍，推測是因為PA4的catechin部位促進了PA4的吸收。然而PA5 (Papp = 2.51 ± 0.21×10-6 cm/s) 卻沒有如同PA4有這麼好的通透率，其可能的原因仍須進一步探究。在各不同比例PA2:EC混合組中，發現EC對於PA2的吸收有干擾作用，造成PA2的通透率減少，各混合組PA2的通透率均約為1.5 × 10-6 cm/s左右，而EC仍無法被吸收。抗發炎實驗中則發現 PA2與其同分異構物及EC均有抑制lipopolysaccharide (LPS) 誘導RAW 264.7巨噬細胞產生NO的效果，其中又以PA2的抑制效果最好；PA2的同分異構物 (PA4及 PA5) 則效果較差；另外，PA2:EC混合組中的效果，則隨著EC所佔比例的增加而降低，推測可能的原因在於EC較不能被細胞吸收所致。抗氧化試驗中發現，EC於各種體外抗氧化試驗中均有較佳的效果；然而在細胞抗氧化試驗 (cell antioxidant activity assay) 則發現，PA2的效果遠優於EC，可能由於EC較不能被細胞吸收所致。Epidemiological studies have shown that dietary flavonoids may contribute to the prevention of oxidative damage in our body. Among different flavonoids, proanthocyanidins have received quite significant interest due to their observed health benefits. Previous study in our laboratory indicated that the water and methanol extracts of longan (Dimocarpus longan Lour.) flower had good antioxidative activity, and proanthocyanidin A2 (PA2) and (-)-epicatechin (EC) were found to be the major active compounds. A2 is a very unstable compound, it could easily be transformed to its isomers epicatechin-(4β→8;2β→O→7)-ent-catechin (PA4) and epicatechin- (4β→6;2β→O→7)- ent-catechin (PA5) in cell culture medium. PA2 and EC are present together in longan flower, so the objective of this study was to use the 4 pure compounds PA2, EC, PA4 and PA5 and also 4 compounds mixtures with different molar or weight ratios of PA2 and EC (2:1), (1:1), (1:2) and nature existence condition (1:3.3), to test their bioavailability, anti-inflammatory and anti-oxidant effects. he Caco-2 cells model has been used to test the bioavailability. Caco-2 cells are derived from human adenocarcinoma and will differentiate into polarized enterocyte-like monolayers, acting similarly to human intestinal epithelial cells. Caco-2 cell monolayer system has provided a useful model to evaluate intestinal transepithelial transport and accumulation of pure phytochemicals. Results of this study showed that PA2 could be absorbed and transported to basolateral side with the apparent permeability coefficient (Papp) of 4.72 ± 0.42×10-6 cm/s but EC could not be transported to basolateral side. On the other hand, PA4 can be absorbed and transport to basolateral side with the Papp = 11.45 ± 5.02×10-6 cm/s which was even higher than PA2, it may be due to the presence of catechin in the lower unit of the molecule. Nevertheless, PA5 (Papp = 2.51±0.21×10-6cm/s) did not possess good permeability as PA4, the reason needs further investigation. As for the mixtures groups, it was found that the permeability of PA2 was interfered by the presence of EC and EC still could not be absorbed by Caco-2 cell. nhibition of LPS-induced NO production in RAW264.7 cell was used as anti-inflammatory assay model. PA2 showed good ability in inhibiting NO formation but PA2 isomers did not show as good effects. Compared to PA2, EC was found to be less effective. As for the mixtures groups, the anti-inflammatory effect was better when EC was in lower ratio in the mixture.n the chemical based in vitro antioxidant assays (DPPH, ORAC) EC had better antioxidant activity than PA2. However, in the cell based antioxidant assay (CAA), the effect of PA2 was far better than EC. The mixture group also has this similarity: when the EC was present more in the ratio, the chemical based in vitro antioxidant effect was better but the cell based antioxidant effect was worse. This may be because EC can not be absorbed by cells.摘要 IBSTRACT III錄 V次 X次 XIII、前言 1、文獻整理 2一節 、體外模式之生物可利用率 2、Caco-2 細胞簡介 2、Caco-2 cell單層膜之模擬動物腸道試驗 4、 Caco-2 cell單層膜完整性及通透率 4二節、發炎 7、發炎簡介 7、巨噬細胞 (Macrophage) 7、一氧化氮(NO) 8、環氧化酵素Cyclooxygenase (COX) 10、脂多醣體Lipopolysaccharide (LPS) 10三節、氧化壓力 12、自由基 12、氧化壓力的主要來源 14、 氧化壓力的測定 15、抗氧化物質 17四節、材料介紹 19、龍眼花 19、原花青素(Proanthocyanidins) 21、兒茶素 22、研究目的與實驗架構 25一節、研究目的 25二節、實驗架構 26、材料與方法 27一節、實驗材料與儀器設備 27、實驗材料 27、實驗細胞株 27、化學藥品與試劑 27一)、化學藥品 27二)、溶劑 29三)、層析材料 29、細胞培養溶液 30、西方墨點法 30、儀器設備 31一)、一般儀器設備 31二)、化學分析儀器設備 31三)、細胞實驗儀器設備 32四)、實驗耗材 33、實驗各種溶液配方 33二節、實驗方法 37、Proanthocyanidin A2及其isomer之分離純化 37一) 龍眼花80%丙酮萃取物之製備 37二) 龍眼花80%丙酮萃取物之溶劑區分萃取 37三) Amberlite XAD-7 resin管柱層析 37四) Sephadex LH-20管柱層析 38五) Proanthocyanidin A2 同分異構物之純化 39六) 高效能液相層析儀 (HPLC) 分析條件 39七) 質譜分析 40、Proanthocyanidin A2及epicatechin之穩定性分析 40一)樣品配製 40二)於細胞通透試驗環境中樣品之穩定性分析 40三) 於細胞抗發炎試驗環境中PA2之穩定性分析 41四) 於細胞抗氧化試驗環境中PA2之穩定性分析 41、以CACO-2细胞模式探討通透率及吸收率 41一) 細胞培養 41二) 細胞繼代培養 41三) Transwell insert 培養 42四) 單層細胞膜完整性測試 42五) Caco-2細胞單層膜通透率試驗 42六) 各樣品經Caco-2細胞單層通透試驗後之分佈 43、以RAW 264.7探討樣品之抗發炎效果 44一) 化學NO (Nitric oxide) 螫合能力測定 44二) 樣品抑制LPS誘導RAW 264.7巨噬細胞NO生成測定 44三) iNOS、COX-2蛋白質表現分析 46、抗氧化實驗 48一) 抑制銅離子誘導人類LDL氧化反應試驗 48二) 清除DPPH自由基活性測定 49三) 氧自由基吸收能力之測驗 (Oxygen radical absorbance capacity, ORAC) 50四) 細胞抗氧化能力測定 (Cellular Antioxidant Avtivity (CAA) assay) 50三節、統計分析 51、結果與討論 53、Proanthocyanidin A2及其isomer之分離純化 53一) 龍眼花80%丙酮萃取物 53二) 龍眼花80 % 丙酮萃取物之溶劑區分萃取 53三) 龍眼花丙酮粗萃物乙酸乙酯層之amberlite XAD-7 resin管柱層析 53四) 龍眼花LF-A-EA-XADII之Sephadex LH-20管柱層析 54五) Proanthocyanidin A2 同分異構物之純化 57、Proanthocyanidin A2及Epicatechin之穩定性分析 63一) PA2於細胞單層膜通透試驗之穩定性分析 63二) PA2於細胞抗發炎試驗之穩定性分析 68三) PA2於細胞抗氧化試驗之穩定性分析 74、以Caco-2细胞模式探討通透率及吸收率 79一) Caco-2細胞存活率分析-MTT assay 79二) 以Caco-2 細胞模式探討待測樣品的吸收率 81.以Caco-2 細胞模式探討EC的吸收率 83以Caco-2 細胞模式探討PA2的吸收率 85. 以Caco-2 細胞模式探討PA4的吸收率 88 以Caco-2 細胞模式探討PA5的吸收率 91 以Caco-2 細胞模式探討PA2:EC(2:1)、(1:1)、(1:2)、(1:3.3) 的吸收率 93、抗發炎實驗 96一) NO (Nitric oxide) 螫合能力測定 96二). 抑制以LPS誘導RAW 264.7 巨噬細胞產生NO之抗發炎效果 98 RAW 264.7細胞存活率分析-MTT assay 98 抑制以LPS誘導RAW 264.7 巨噬細胞產生NO之抗發炎效果 100三) . iNOS、COX-2蛋白質表現分析 102、抗氧化實驗 103一) DPPH自由基清除能力 103二) 氧自由基吸收能力之測驗 (Oxygen radical absorbance capacity, ORAC) 105三) 抑制銅離子誘導人類LDL氧化反應試驗 108四) 細胞抗氧化模式 110 細胞存活率分析-MTT assay 110 細胞抗氧化能力測定 (Cellular Antioxidant Activity (CAA) assay) 112、結論 122. 穩定性試驗 122、參考文獻 124、附錄 13