Bioavailability and metabolism of botanical constituents and enhancement of intestinal barrier function by caffeic acid derivatives in Caco-2 cells.

摘要

Public interest in botanical supplements has increased greatly in recent years as various plant materials might be used for anti-inflammatory, immunostimulatory, antioxidant and cancer preventive effects. Our long term goal is to improve our understanding of the characteristics of phytochemicals that contribute to human health benefits on gut functions, and thereby pave the way for optimizing herbal supplements for study in future clinical trials. In this dissertation, the overarching hypotheses were that major components of the ethanolic extracts of Echinacea, alkamides and ketones, and caffeic acid derivatives in the ethanolic extract of Prunella vulgaris, will be transferred by Caco-2 cell monolayers and caffeic acid derivatives will enhance the intestinal epithelial barrier function.;Echinacea has long been used as phytotherapy for wound healing, pain relief and treatment of the common cold. In the first study, Bauer alkamides, the key components contained in Echinacea sanguinea and Echinaceapallida, transferred across the Caco-2 cell monolayer via passive diffusion, independent of other constituents in plant extract. The apparent permeability coefficients (Papp) were 2.8 +/- 1.5- 43.8 +/- 11.2 cm/s x10-6 for tested three alkamides and the order of the transfer of them across Caco-2 cells was increased paralleled with compound hydrophicility. Tested alkamides were seemingly N-glucuronidated and both Echinacea extracts stimulated apparent glucuronidation and basolateral efflux of alkamide metabolites. Bauer ketone 24 was totally metabolized to more hydrophilic metabolites as a pure compound, but not found in either Echinacea species. The addition of Bauer alkamides (175-230 microM) as well as the ethanolic extracts of E. sanguinea at 1 mg/mL (containing 85 microM of alkamide 8, 2 microM of alkamide 10, and 0.7 microM of alkamide 11) and E. pallida at 5 mg/mL (containing 215 microM of alkamide 8, 25 microM of alkamide 10, and 45 microM of alkamide 11) reduced the efflux of the P-glycoprotein transporter (P-gp) probe calcein-AM from Caco-2 cells. These results suggest that other constituents in the plant extract had a facilitating effect on the metabolism and efflux of alkamides and ketones from Echinacea , which would improve the therapeutic benefits of these extracts, and that alkamides and Echinacea extracts might be useful in potentiating some chemotherapeutics which are substrates for P-gp.;Prunella vulgaris is a perennial herb known as self-heal used to treat sore throat, fever, and wounds. Rosmarinic acid is a caffeic acid derivative found in various botanicals, especially in P. vulgaris. Ursolic acid, a pentacyclic triterpene acid, is also found in P. vulgaris but especially concentrated in Salvia officinalis (sage), which has been traditionally used to treat inflammation in the oral cavity, and may also be of interest in inhibiting gastrointestinal inflammation which is relevant to colitis and colon cancer. In the second study, Papp for rosmarinic acid and rosmarinic acid in P. vulgaris extracts was 0.2 +/- 0.05 x 10-6 cm/s, significantly increased to 0.9 +/- 0.2 x 10-6 cm/s after beta-glucuronidase/sulfatase treatment. Papp for ursolic acid and ursolic acid in S. officinalis extract was 2.7 +/- 0.3 x 10-6 cm/s and 2.3 +/- 0.5 x 10-6 cm/s before and after beta-glucuronidase/sulfatase treatment, respectively. Neither compound was affected in permeability by the herbal extract matrix. These results indicate that rosmarinic acid and ursolic acid in herbal extracts had similar uptake as that found using the pure compounds, which may simplify the prediction of compound efficacy, but the apparent lack of intestinal glucuronidation/sulfation of ursolic acid is likely to further enhance the bioavailability of that compound compared with rosmarinic acid.;In the third study, the effects of caffeic acid and related compounds on intestinal barrier function were investigated using Caco-2 cells as a model. Caffeic acid, rosmarinic acid, chlorogenic acid and m-hydroxyphenylpropionic acid (mHPP, a microbial metabolite of caffeic acid and rosmarinic acid) up-regulated the expression of zonula occludens (ZO -1), ZO-2, claudin-1 and occludin in Caco-2 cells. In addition, chlorogenic acid and mHPP were effective against adverse effects induced by inflammatory stimuli (LPS, interferon-gamma, IL-1beta, and TNF-&agr;) on tight junction proteins in Caco-2 cells. Caffeic acid derivatives up-regulated claudin-4 in P-glycroprotein transporter MDR1- knockdown (KD) Caco-2 cells and only mHPP was effective against the changes in tight junction protein expression induced by inflammatory stimuli in MDR KD Caco-2 cells. Caffeic acid derivatives augmented TNF-&agr; and IL-6 levels in Caco-2 cells under the stimulated condition, but significantly reduced both cytokines in MDR KD caco-2 cells plus the stimuli. These results indicate that caffeic acid derivatives enhanced barrier function in human intestinal Caco-2 cells and mHPP exhibited greater enhancement of intestinal barrier than the parent compounds. P-gp plays an essential role in the anti-inflammatory activities of caffeic acid derivatives.;In conclusion, these data confirmed the overarching hypotheses and suggest that the effect of plant matrix on bioavailability and metabolism of the constituent is compound specific, depending on the transfer mechanism; and caffeic acid derivatives could be gut health promoting as a dietary constituent, but these compounds might exacerbate damage under inflammatory stimuli. Moreover, intake of caffeic acid derivatives might speed up mucosal recovery or provide protection to the small-intestinal mucosa against the inflammatory mediators when P-gp inhibitors are co-administered, which may be attractive from a therapeutic point of view.