Intestinal permeability and gut microbiota interactions of phytochemicals from herbal drugs used in the treatment of depressive, anxiety and sleeping disorders

The use of herbal medicines to treat depression, anxiety and sleeping disorders is a tendency that has increased over the past decades. Valerian (Valeriana officinalis L., Caprifoliaceae), St. John’s wort (Hypericum perforatum L., Hypericaceae) and California poppy (Eschscholzia californica, Cham., Papaveraceae) are herbal medicines that are commonly used for the management of these disorders. They contain pharmacologically relevant compounds such as valerenic acid (valerian), hyperforin and hypericin (St. John’s wort), and californidine, escholtzine and protopine (California poppy). To date, the intestinal permeability of these herbal compounds has been poorly investigated. In addition, the reciprocal interactions between these compounds and the human gut microbiota have not been explored; in particular, their possible biotransformation by the gut microbiome, or a possible impact of the exposure of these herbal compounds or extracts on the microbial metabolism of short-chain fatty acids (SCFAs) and on the bacterial viability. In light of the growing number of studies describing the biotransformation of oral compounds (drugs, dietary compounds, or phytochemicals) by the gut microbiome, or the emerging evidence that gut microbial metabolites such as SCFAs play a significant role in the modulation of the central nervous system (CNS) via the microbiota-gut-brain axis, an assessment of these gut microbiota interactions is necessary. Finally, considering herbal medicines, unlike for valerian and St. John’s wort, only very few studies have determined the contents of pharmacologically active constituents in commercial preparations of California poppy (californidine, escholtzine and protopine), making it challenging to estimate the levels that can be anticipated in patients.
Firstly, the intestinal permeability of herbal compounds valerenic acid, hyperforin, hypericin, californidine, escholtzine and protopine was studied, with filter-grown Caco-2 cell monolayers as a model of the human small intestinal epithelium. The model was validated with markers of low-to-moderately and highly permeable compounds, atenolol and propranolol, respectively. The transport of all compounds was assessed in both directions, from apical to basolateral and vice versa.
Thereafter, the gut microbiota interactions between herbal extracts and compounds were evaluated with short-term batch fermentation experiments (24 h) using an in vitro gut microbiota, the Polyfermentor Intestinal Model (PolyFermS). Experiments were conducted with effluents from two PolyFermS, each one generated from a different healthy fecal donor. The validity of both PolyFermS was confirmed by stable gut microbiota composition and steady metabolic activity (production of SCFAs), hallmarks of a healthy gut microbiome. Batch fermentation experiments allowed, on the one hand, the assessment of a microbiota-mediated biotransformation of herbal compounds, and on the other hand, the measurement of microbial metabolic activity (SCFAs production) and bacterial viability in the presence of herbal compounds and extracts. SCFAs were quantified by HPLC coupled with a refractive index detector and total viable and dead bacterial cells were determined by flow cytometry.
Furthermore, the contents of californidine, escholtzine and protopine were determined in eight commercial preparations of California poppy, sold as phytomedicines or food supplements. The preparations consisted of dry herbal powders, dry extracts or fluid extracts of flowering aerial parts of California poppy.
For each herbal compound, a sensitive and selective UHPLC-MS/MS method was developed and validated for the analysis of Caco-2 cells, gut microbiota and California poppy samples.
Valerenic acid, hyperforin, escholtzine and protopine were highly absorbed in the Caco-2 cell model. A carrier-mediated process was possibly involved in the transport of valerenic acid (uptake), escholtzine (efflux) and protopine (efflux), whereas hyperforin was likely transported by passive diffusion. In addition, the low recovery values (13 – 69%) obtained for valerenic acid, escholtzine and protopine suggest that the compounds might be metabolized in the Caco-2 cells. In turn, hypericin and californidine showed a low-to-moderate absorption, possibly with passive diffusion as transport process for hypericin and active transport for californidine (efflux). These data indicate that hypericin and californidine have the potential to be found at relevant concentrations in the colon segment, where the gut microbiome is the densest.
All the herbal compounds showed a high stability in the batch fermentation experiments, suggesting that they are not biotransformed by the human gut microbiome. These results support that the disposition of these herbal compounds is not influenced by the gut microbiome. Furthermore, the exposure of herbal extracts and compounds did not markedly impact the bacterial metabolism of SCFAs or the bacterial viability. This suggests that valerian, St. John’s wort or California poppy, at the tested concentrations, may not exert an indirect effect on the CNS via modulation of bacterial SCFAs implicated in microbiota-gut-brain axis signaling, at least not after a short-term exposure (24 h). Given the high inter-individual variabilities between human gut microbiota and short-term experiments setup, these findings need to be confirmed with a larger number of microbiota and with continuous fermentation models to further evaluate the possible impact on microbiota metabolic activity and bacterial viability, over a prolonged exposure.
Alkaloid contents differed strongly between the commercial products of California poppy, ranging from 0.13 – 2.55 mg/g for californidine, 0.05 – 0.63 mg/g for escholtzine and 0.008 – 0.200 mg/g for
protopine. These marked variations are likely due, at least in part, to differing extraction procedures.
Based on these data and the dosage recommended by manufacturers, maximum daily doses for the three alkaloids were calculated to range from 0.16 to 2.97, 0.10 to 1.11, and 0.02 to 0.31 mg/day, respectively. Thus, for patients using different California poppy preparations, significant variations in the daily intake of californidine, escholtzine and protopine, have to be expected.