Kerry Penman

Permeability studies of alkylamides and caffeic acid conjugates from echinacea using a Caco-2 cell monolayer model

Background:  Echinacea is composed of three major groups of compounds that are thought to be responsible for stimulation of the immune system – the caffeic acid conjugates, alkylamides and polysaccharides. This study has focussed on the former two classes, as these are the constituents found in ethanolic liquid extracts.

Steroidal saponins from the roots of Trillium erectum (Beth root).

Eleven steroidal saponins including three previously unreported saponins 1-3, two known ecdysteroids and one fatty acid, have been isolated from the roots of Trillium erectum (Beth root) by RP-HPLC and characterized by spectroscopic (1D and 2D NMR experiments) and spectrometric (LCMS) methods.

Modulation of macrophage immune responses by Echinacea

Echinacea preparations are widely used herbal medicines for the prevention and treatment of colds and minor infections. There is little evidence for the individual components in Echinacea that contribute to immune regulatory activity. Activity of an ethanolic Echinacea extract and several constituents, including cichoric acid, have been examined using three in vitro measures of macrophage immune function – NF-κB, TNF- α and nitric oxide (NO). In cultured macrophages, all components except the monoene alkylamide (AA1) decreased lipopolysaccharide (LPS) stimulated NF-κB levels. 0.2 µg/ml cichoric acid and 2.0µg/mL Echinacea Premium Liquid (EPL) and EPL alkylamide fraction (EPL AA) were found to significantly decrease TNF-α production under LPS stimulated conditions in macrophages. In macrophages, only the alkylamide mixture isolated from the ethanolic Echinacea extract decreased LPS stimulated NO production. In this study, the mixture of alkylamides in the Echinacea ethanolic liquid extract did not respond in the same manner in the assays as the individual alkylamides investigated. While cichoric acid has been shown to affect NF-κB, TNF-α and NO levels, it is unlikely to be relevant in the Echinacea alterations of the immune response in vivo due to its non- bioavailability – i.e. no demonstrated absorption across the intestinal barrier and no detectable levels in plasma. These results demonstrate that Echinacea is an effective modulator of macrophage immune responses in vitro.

Bioavailability of Echinacea constituents: Caco-2 monolayers and pharmacokinetics of the alkylamides and caffeic acid conjugates.

Many studies have been done over the years to assess the effectiveness of Echinacea as an immunomodulator. We have assessed the potential bioavailability of alkylamides and caffeic acid conjugates using Caco-2 monolayers and compared it to their actual bioavailability in a Phase I clinical trial. The caffeic acid conjugates permeated poorly through the Caco-2 monolayers. Alkylamides were found to diffuse rapidly through Caco-2 monolayers. Differences in diffusion rates for each alkylamide correlated to structural variations, with saturation and N-terminal methylation contributing to decreases in diffusion rates. Alkylamide diffusion is not affected by the presence of other constituents and the results for a synthetic alkylamide were in line with those for alkylamides found in an ethanolic Echinacea preparation. We examined plasma from healthy volunteers for 12 hours after ingestion of Echinacea tablets manufactured from an ethanolic liquid extract. Caffeic acid conjugates could not be identified in any plasma sample at any time after tablet ingestion. Alkylamides were detected in plasma 20 minutes after tablet ingestion and for each alkylamide, pharmacokinetic profiles were devised. The data are consistent with the dosing regimen of one tablet three times daily and supports their usage as the primary markers for quality Echinacea preparations.

Polyunsaturated alkyl amides from Echinacea: synthesis of diynes, enynes, and dienes.

The synthesis of 20 alkyl amides, including 15 naturally occurring polyunsaturated alkyl amides previously identified from Echinacea spp. (1-13 and 62) or from Achilla sp. (55) and five previously unknown geometric isomers (23, 28, 67, 73, and 80), is described. Importantly, these amides include all of the major alkyl amides present in commercially used Echinacea extracts. The syntheses demonstrate methodology used for constructing alkyl amides containing conjugated diyne and isomerically pure enyne and diene moieties and may be adapted easily for the preparation of other alkyl amides present in Echinacea spp. Terminal-conjugated diynes were prepared by a Cadiot-Chodkiewitz coupling/deprotection sequence utilizing a protected bromoacetylene, and methyl-substituted diynes were made via a base-catalyzed rearrangement of terminal-skipped diynes. Conjugated dienes were prepared conveniently and with high stereoselectivity by the reduction of enynes or diynes with Rieke zinc. With the exception of 1-2 and 11-12, the alkyl amides are synthesized here for the first time, and their NMR data are consistent with that of the reported isolated natural compounds.

Allyl- and allyllike-tin derivatives: II. The crystal structure of cyclopent-2-enyltriphenyltin☆

Abstract The crystal structure of (C 6 H 5 ) 3 Sn(cyclopent-2-enyl) has been determined. The asymmetric unit consists of two independent molecules of cyclopent-2-enyltriphenyltin, in which the Ph 3 Sn groups are bonded to the C 5 rings in an axial manner. An interesting feature is the diastereoisomeric relation between the two molecules: the phenyl groups have opposite paddle-wheel configurations with respect to the fixed chirality of the C 5 ring carbon atom bonded to tin. The geometrical parameters, establishing a preferred quasi- axial orientation for the SnPh 3 group, exclude p π  d π bonding between the metal and double bond; and may reflect a ground state stabilising effect (of the σ-π hyperconjugative type) that operates more efficiently from this orientation. A similar phenomenon was suggested to operate in the acyclic Ph 3 SnCH 2 CHCH 2 on the basis of its crystal structure.

Permeability studies of Kavalactones using a Caco‐2 cell monolayer model

Objective:  To examine the bioavailability of kavalactones in vitro and the possible differences in their bioavailability because of variations in either chemical structure or the method of extraction used.

13C chemical-shift assignments in cyclooctyl derivatives from the spectra of deuterioisotopomers. Deuterium isotope effects on chemical shifts and conformational equilibria

The 13C NMR spectra of some derivatives of (Z)-cyclooctene, cyclooctanols and cyclooctanone have been assigned by consideration of substituent effects, 1H–13C correlated spectra, and 2H isotope effects. Some four-bond downfield 2H effects (+4Δ) on certain chemical shifts have been measured and attributed to conformational equilibrium perturbations and trans-annular 1H–1H interactions. The data provide a basis for further assignments in these systems.

Stereochemistry of acidolysis of cyclohept-2-enyl-silanes and -stannanes

Direct 2H n.m.r. analysis of the 2H-substituted 3- and 4-methylcycloheptenes produced by acid cleavage (CF3CO2D) of mixtures of 4- and 7-methylcyclohept-2-enyltrimethyl-silanes and -stannanes confirms stereospecific γ-anti attack by the electrophile.