Metabolomic analysis of Ranunculus spp. as potential agents involved in the etiology of equine grass sickness.

Identification of toxic or harmful agents continues to be a key goal in agricultural chemistry. This paper reports a metabolomic analysis of Ranunculus repens and related species, which were recently postulated to be cocausative agents in the etiology of equine grass sickness (EGS). Specifically, samples collected at EGS sites were compared with those from non-EGS sites. Furthermore, interspecific and seasonal variations and the species' response to edaphic and climatic factors were investigated. (1)H NMR spectroscopy in combination with multivariate data analysis was applied to the crude methanol extracts of the Ranunculus samples, as well as their chloroform fractions. Samples from EGS sites were significantly different from control samples. The metabolite composition varied greatly between different Ranunculus species. No significant changes could be observed between samples collected in different seasons. This work provides strong evidence that Ranunculus is involved in the etiology of EGS and has implications for agricultural management of pastures.

The interaction potential of herbal medicinal products: a luminescence-based screening platform assessing effects on cytochrome P450 and its use with devil's claw (Harpagophyti radix) preparations.

OBJECTIVES: Potential interactions between herbal medicinal products and the cytochrome (CYP) P450 system are an important safety concern. We set out to develop a screening panel for assessing such interactions and use it to evaluate the interaction potential of devil's claw. METHODS: The panel consisted of luminescence-based inhibition assays for CYP1A2, 2C9, 2C19, 2D6 and 3A4, and a reporter gene (luciferase) assay for pregnane X receptor (PXR) activation and CYP3A4 induction. Caftaric acid and chlorogenic acid, two compounds with strong fluorescence quenching properties, were used to demonstrate the assay's resistance to interference. We tested 10 commercial devil's claw preparations as well as harpagoside and harpagide, two important constituents of devil's claw. KEY FINDINGS: Five preparations were found to weakly inhibit CYP3A4 (IC50 124.2-327.6 µg/ml) and five were found to weakly activate PXR (EC50 10.21-169.3 µg/ml). Harpagoside and harpagide did not inhibit CYP3A4. In agreement with published data, bergamottin, a natural product known to interact with CYP3A4, was shown to inhibit CYP3A4 with an IC50 of 13.63 µm and activate PXR with an EC50 of 6.7 µm. CONCLUSIONS: Devil's claw preparations are unlikely to have a clinically relevant effect on CYP function. The assay panel proved effective in screening devil's claw preparations, demonstrating its suitability for use with plant extracts. It showed superior sensitivity and resistance to interference.

Safety of Herbal Medicinal Products: Echinacea and Selected Alkylamides Do Not Induce CYP3A4 mRNA Expression.

A major safety concern with the use of herbal medicinal products (HMP) is their interactions with conventional medicines, which are often mediated via the cytochrome P450 (CYP) system. Echinacea is a widely used over-the-counter HMP, with proven immunomodulatory properties. Its increasing use makes research into its safety an urgent concern. Previously, we showed that Echinacea extracts and its alkylamides (thought to be important for Echinacea's immunomodulatory activity) mildly inhibit the enzymatic activity of the main drug metabolising CYP isoforms, but to this date, there is insufficient work on its ability to alter CYP expression levels. We now report for the first time the effect of a commercial Echinacea extract (Echinaforce) and four Echinacea alkylamides on the transcription of the major drug metabolizing enzyme CYP3A4. HepG2 cells were exposed for 96 h to clinically relevant concentrations of Echinaforce (22, 11.6 and 1.16 µg mL(-1)) or the alkylamides (1.62 and 44 nM). CYP3A4 mRNA levels were quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Neither Echinaforce nor the alkylamides produced any significant changes in the steady-state CYP3A4 mRNA levels, under these conditions. In contrast, treatment with 50 µM rifampicin resulted in a 3.8-fold up-regulation over the vehicle control. We conclude that Echinaforce is unlikely to affect CYP3A4 transcriptional levels, even at concentrations which can inhibit the enzymatic activity of CYP3A4. Overall, our data provides further evidence for the lack of interactions between Echinacea and conventional drugs.

Metabolomic profiling of liquid Echinacea medicinal products with in vitro inhibitory effects on cytochrome P450 3A4 (CYP3A4).

ECHINACEA is a popular and widely used herbal medicinal product and consequently, studies of its interactions with conventional drugs are of particular importance. We have shown that ECHINACEA preparations and some common alkylamides weakly inhibit several cytochrome P450 (CYP) isoforms, with considerable variation in potency. We now report a detailed analysis of six commercial ECHINACEA liquid preparations, with emphasis on the metabolomic characterisation of the ECHINACEA compounds responsible for inhibiting CYP3A4. We separated each preparation into its ethanol- and water-soluble components, and then used (1)H-NMR together with multivariate data analysis and partial least square regression analysis to investigate the nature of the compounds responsible for CYP3A4 inhibition. The results implicated alkylamides in the CYP3A4 inhibitory activity of ECHINACEA. One of the commercial preparations (Echinaforce(R)) was further fractionated using solid phase extraction. Analysis by (1)H-NMR and mass spectroscopy (LC/MS, tandem MS, accurate mass) identified dodeca-2 E,4 E,8 Z,10 E/Z-tetraenoic acid (alkylamide 1) and a new compound (putative molecular formula C (18)H (36) NO (+)) as major components of the inhibitory fractions. In addition, the alkylamide content of all six preparations was determined by reverse phase HPLC. Levels of alkylamides 1 and 3 (undeca-2 E,4 E/ Z-diene-8,10-diynoic acid isobutylamide), correlated well with CYP3A4 inhibition. The acetylene tetradeca-8 Z-ene-11,13-diyn-2-one was shown to be present in the E. PURPUREA as well as the E. PALLIDA extracts. E. PURPUREA unlike E. PALLIDA was thought to not contain significant amounts of acetylenes. Our results directly confirm the role of alkylamides in the inhibition of CYP3A4 by ECHINACEA and uncovered a new compound which may also be involved. Extensive differences in the composition of the commercially available preparations were found. This will inevitably impact on the product efficacy, safety and pharmacological effects, especially since the differences involve alkylamides, an important class of ECHINACEA's active constituents. The metabolomic approach presented here may prove valuable as a screening or quality control tool.

Herbal extracts used for upper respiratory tract infections: are there clinically relevant interactions with the cytochrome P450 enzyme system?

The regulatory requirements for assessing potential interactions between herbal medicinal products and other medicines can cause specific, additional phytopharmaceutical problems. In this short review we assess the state of our knowledge for herbal extracts commonly used as over the counter (OTC) products for upper respiratory infections and which in many cases are considered to have immunomodulatory effects. Overall, the data on the safety of these products is still limited and only in the case of Echinacea preparations can preliminary conclusions be drawn. The available evidence points to weak cytochrome P450 inhibition which is unlikely to be of clinical relevance.