Transport Mechanism Of Diterpenoids From Andrographis Paniculata In Caco-2 Monolayers

Objective:The present study was to investigate the absorptive mechanism of andrographolide, dehydroandrographolide and neoandrographolide and to refer the experimental and theories gist for a deep study of the pharmacokinetics and pharmacological mechanism, using Caco-2 cells as models and determing apparent octanol-water partition coefficient.Methods:①The apparent octanol-water/buffer partition coefficient of andrographolide, dehydroandrographolide and neoandrographolide was measured by shaking flask method.②An LC/MS/MS method was developed for analyzing the Caco-2 cell assay samples. Establish and assess the characteristic of Caco-2 cell monolayer. Caco-2 cell monolayer model was used to investigate the bidirectional transport of andrographolide, dehydroandrographolide and neoandrographolide. The effect of time, drug concentration, system temperature and inhabitors on the absorption of them was observed for exploring the absorptive characteristic and transcellular efflux.③The transport of andrographolide and neoandrographolide was studied while two compound were co-administrated. The effect of neoandrographolide on transport of andrographolide was tested across Caco-2 cell monolayers.Results:①The apparent octanol-water partition coefficient (Papp) of andrographolide, dehydroandrographolide and neoandrographolide were 3.90 (log Papp=0.29),19.75 (log Papp=1.30) and 1.75 (log Papp=0.24) in water at 37℃respectively. Andrographolide and neoandrographolide have the highest partition coefficient in pH6, and dehydroandrogapholide in pH5.②Caco-2 cells cultured on transwell plate had formed a tight structure with a TEER value of Caco-2 monolayers being greater than 500Ω·cm2. Permeability and the expression of alkaline phosphatase in the cell model were superior.②Time and concentration saturation were observed for the absorptive transport of andrographolide across Caco-2 monolayers. The transport of andrographolide was influenced by the change of temperature and the presence of iodoacetamide, but not Verapamil or MK-571.④The amount of dehydrographolide which was transported increased linearly with the time and concentration. And it was not influenced by the change of temperature and the presence of Verapamil or MK-571.⑤The amount of dehydrographolide which was transported increased linearly with the time and concentration. But it was influenced by the change of temperature and the presence of iodoacetamide, but not Verapamil or MK-571.⑥Co-administration of andrographolide and neoandrographolide leaded to a decrease transport of andrographolide from apical side to basolateral side when concentration of neoandrographolide increased.Conclusions:①The Papp of andrographolide, dehydroandrographolide and neoandrographolide was influenced by pH, and the higher pH could decrease Papp of them.②The study established an integrated Caco-2 cell monolayers model, which met the requirement of the drug transport experiment.③The absorption and transport of andrographolide in Caco-2 cell monolayer was an active transportation mediated by transporter, which mainly located in the apical side of Caco-2 cell monolayers.④The absorption and transport of dehydroangrapholide was passive diffusion as the dominating process in Caco-2 cell monolayers.⑤The absorption and transport of dehydroangrapholide was passive diffusion and active transport. And the transporter maybe located in the apical side of Caco-2 cell monolayers.⑥The absorption and transport of andrographolide could be inhibited by neoandrographolide. They may be mediated by the same transporter.