Study On The Intestinal Absorption Of The Chemical Components Of Trollius Based On The Caco-2 Cell Model

Although the chemical components and in vitro activites of the flowers of T.chinensis are very clear,the effective components in these flowers are still controversial.The key to solve above controversy is to reveal their in vivo behaviors.Absorption,distribution,metabolism and excretion are the main in vivo process of the flowers,and absorption by intestine is very crucial to in vivo activites of these flowers for its influence on another process.Therefore,it is necessary to study the intestinal absorption of the flowers of T.chinensis for the evaluation oftheir effective components.Caco-2 cell monolayer model,one of ADME/Tox typical methods,was applied to investigate the intestinal absorption of twelve compounds,including four phenolic acids(veratric acid,proglobeflowery acid,globeflowery acid and trollioside),seven flavonoids(orientin,vitexin,2?-O-?-L-galactopyranosylorientin,2?-O-?-L-galactopyranosylvitexin,isoswertisin,isoswertiajaponin,and trollisin ?),and one alkaloid(trolline),and crude extract.The effects of incubating time and concentration on the transport of twelve compounds were studied,and P-gp inhibitor(verapamil hydrochloride)was employed in the transport experiment to determine whether compounds(orientin,vitexin,2?-O-?-L-galactopyranosylorientin,and 2?-O-?-L-galactopyranosylvitexin)were exported by P-gp.Meanwhile,the permeability was predicted according to drug-likeness assessed with software,and the relationship between intestinal absorption and molecular structure was also investigated.Moreover,broth microdilution method was applied to test the minimal inhibitory concentration(MIC)of crude extract and mixture of absorbed components against Gram-positive(Staphylococcus aureus)and Gram-negative bacteria(Pseudomonas aeruginosa,Proteus vulgarb and subsp.pneumoni),and Griess assay was used to investigate the inhibitory effect of crude extract and mixture of absorbed components against LPS induced production of NO in RAW 264.7 cells.Anti-bacterial activity and anti-inflammatory activity of crude extract and mixture of absorbed components were comparatively evaluated by MIC values and the amount of NO,respectively.The results were as follows.Phenolic acids were well absorbed through passive diffusion,and the molecular polarity had a great effect on the absorption.Flavonoids were poorly absorbed,and they were mediated via a passive diffusion mechanism primarily,coexisting with a carrier-mediated transport.Orientin and vitexin were exported by P-gp in the AP side.Although 2?-O-?-L-galactopyranosylorientin and 2?-O-?-L-galactopyranosylvitexin were not the substrates of P-gp,they might be exported by another efflux protein.Another three flavonoids(isoswertisin,isoswertiajaponin and trollisin ?)were transported through passive diffusion.Alkaloid(trolline)was moderately absorbed through an associative mechanism involving active and passive transport,and more research is needed to confirm the detailed transport mechanism.Veratric acid,proglobeflowery acid,trollioside,orientin,vitexin,2?-O-?-L-galactopyranosylorientin and 2?-O-?-L-galactopyranosylvitexin were the main permeable coruponents in the crude extract.The proportion of phenolic acids in the crude extract increased from about 6.82%to 61.18%,while that of flavonoids decreased from 93.18%to 38.82%after passing through Caco-2 cell monolayer.Anti-bacterial and anti-inflammatory experiments showed that the mixture of absorbed components had stronger anti-bacterial and anti-inflammatory activities than crude extract.From the above results,we can conclude that phenolic acids,flavonoids and trolline in the flowers of T.chinensis were well,poorly and moderately absorbed,respectively.After the crude extract was absorbed,its composition changed dramatically.Phenolic acids,the minor components in the crude extract,became the dominant part of the absorbed compounds,while flavonoids declined from the dominant part to subordinative part.Flavonoids and phenolic acids were the effective anti-bacterial components,and phenolic acids were the main anti-inflammatory component in the flowers of T.chinensis.This study further undermined the traditional view that flavonoids are the main effective components of the flowers of T.chinensis,and led us clearly understand the major contributors to the efficacy of these flowers.