以體外Caco-2 細胞模式系統探討龍眼花中proanthocyanidin A2 及epicatechin之生物可利用率以及其抗發炎與抗氧化效果

Abstract

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。 本研究利用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。 Caco-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。 在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。 在抗氧化試驗中發現，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. PA2 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. The 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. Inhibition 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. In 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.