生姜鲜榨汁中纳米囊泡的离体肠吸收特点及体外吸收机制研究

Intestinal absorption characteristics and absorption mechanism in vitro of nano vesicles from fresh Zingiber officinale juice

目的 阐明生姜Zingiber officinale鲜榨汁纳米囊泡结构对其中成分离体肠吸收的影响及其被肠细胞摄取的机制。方法 富集生姜中的纳米囊泡进行冻干处理,复溶后对其进行表征;同时,超声制备的纳米囊泡破碎组样品,采用液质联用技术对二者肠吸收液中的6种姜辣素类成分进行测定,计算各成分在不同肠段中的累积吸收量、吸收速率常数及表观渗透系数。通过CCK-8法测定纳米囊泡对人结直肠腺癌Caco-2细胞的细胞毒性;利用PKH67、鬼笔环肽和DAPI分别对纳米囊泡、Caco-2细胞的细胞骨架及细胞核进行标记,观察细胞对纳米囊泡的摄取情况;加入不同类型的摄取抑制剂,采用流式细胞术检测荧光强度,探究纳米囊泡的细胞摄取机制。结果 生姜鲜榨汁纳米囊泡冻干粉得率为2.21%;纳米囊泡组肠吸收样品中未检测到6-去氢姜二酮(6-dehydrogingerdione,6-De),纳米囊泡破碎组肠吸收样品中未检测到6-De和10-姜酚(10-gingerol,10-Gi);纳米囊泡组4-Gi和6-姜烯酚(6-shogaol,6-Sh)在各肠段中的表观渗透系数均高于纳米囊泡破碎组(P<0.05);确定纳米囊泡冻干粉质量浓度为500μg/mL进行PKH67荧光染色,加入阿米洛利与甲基-β-环糊精抑制剂的2组细胞荧光强度分别下降了26%、30%。结论 纳米囊泡可促进生姜中成分的吸收,且可被Caco-2细胞完整摄取,其可能通过巨胞饮途径和脂筏介导途径进入细胞。

Objective To clarify the impact of the structure of plant-derived nanovesicles in Shengjiang(Zingiber officinale)juice on the intestinal absorption of its internal constituents,and to explore the uptake mechanism in vitro.Methods First,enrich the nanovesicles followed by freeze-drying,and the freeze-dried powder was rehydration,the nanovesicles fragmentation samples were prepared by ultrasound,and six gingerol compounds in both intestinal sac absorption fluid were determined by LC-MS/MS method.The actual cumulative absorption quantity per unit area,the absorption rate(Ka)and apparent permeability coefficient(Papp)were calculated and compared between two groups.The cytotoxicity of nanovesicles to Caco-2 cells was determined using the CCK-8 method to determine appropriate concentration.PKH67,phalloidin and DAPI were used to label nano-vesicles and the cytoskeleton and nucleus of Caco-2 cells respectively,and the uptake of nanocapsules by cells were observed.The uptake mechanism of nanovesicles was studied using flow cytometry after adding different types of uptake inhibitors.Results The yield of freeze-dried powder from Z.officinale juice nanovesicle was 2.21%.6-dehydrogingerdione(6-De)was not detected in nanovesicle group,and 6-De and 10-gingerol(10-Gi)were not detected in the disrupted nanovesicle group.The Papp of 4-Gi and 6-shogaol(6-Sh)were higher in the nanovesicle group than those in disrupted nanovesicle group in all four intestinal segments with statistical significance(P<0.05).500μg/mL freeze-dried powder nanocapsules was fluorescence stained with PKH67 to observe the uptake results,the fluorescence intensity of amiloride group and methyl-β-cyclodextrin group decreased by 26%and 30%respectively.Conclusion The nanocapsules can promote the absorption of the main components in Z.officinale,and nanocapsules can be uptaken with a complete structure by Caco-2 cells,the results indicate through the raft-mediated pathway and macropinocytosis pathway.