Alkaloids from Lime Flower (Tiliae flos) Exert Spasmodic Activity on Murine Airway Smooth Muscle Involving Acetylcholinesterase.

Lime flower (Tiliae flos) is traditionally used either for treatment of the common cold or to relieve symptoms of mental stress. Recently, the presence of a new class of piperidine and dihydro-pyrrole alkaloids from lime flower has been described. The present study aimed to investigate the pharmacological activity of hydroacetonic lime flower extracts, alkaloid-enriched lime flower fractions, and isolated alkaloids on the murine airway smooth muscle and the cholinergic system. While a hydroacetonic lime flower extract did not show any pharmacological activity, enriched Tilia alkaloid fractions potentiated acetylcholine-induced contractions of the trachea by ~ 30%, showing characteristics comparable to galanthamine. Effects were abrogated by atropine, indicating an involvement of muscarinic receptors. The dihydro-pyrrole alkaloid tiliine A, the piperidine alkaloid tiliamine B, and the acetylated piperidine alkaloid tilacetine A were characterized as acetylcholinesterase inhibitors. The positive control galanthamine (IC50 = 2.0 µM, 95% CI 1.7 to 2.2 µM) was approximately 100 times more potent compared to tiliine A (IC50 = 237 µM, 95% CI 207 to 258 µM) and tiliamine B (IC50 = 172 µM, 95% CI 158 to 187 µM). Neither DNA synthesis of HepG2 liver cells, HaCaT keratinocytes, and Caco-2 intestinal epithelial cells nor cell viability of primary human fibroblasts was reduced by the alkaloids. The indirect cholinergic activity of the alkaloids might explain some aspects of the traditional use of lime flowers and may extend the portfolio of compounds with regard to diseases involving parasympathetic malfunction or central cholinergic imbalance.

Cryptotanshinone from Salvia miltiorrhiza Roots Reduces Cytokeratin CK1/10 Expression in Keratinocytes by Activation of Peptidyl-prolyl-cis-trans-isomerase FKBP1A.

Cryptotanshinone (CTS) (1 µM) from the roots of Salvia miltiorrhiza exerts a strong influence on the terminal differentiation of human keratinocytes (HaCaT cell line, primary natural human keratinocytes) and downregulates the expression of differentiation-specific cytokeratins CK1 and CK10 on protein and gene level. Other differentiation specific proteins as involucrin, filaggrin, loricrin, and transglutaminase were not affected to a higher extent. CTS (1 µM) did not influence the cell viability and the proliferation of keratinocytes. Using a combination of drug affinity response target stability assay in combination with a proteomic approach and multivariate statistics for target elucidation, peptidyl-prolyl-cis-trans-isomerase FKBP1A (known target of inhibitors such as tacrolimus or rapamycin) was addressed as potential molecular target of CTS. The interaction of CTS with FKBP1A was additionally shown by thermal shift and enzymatic activity assays. Interestingly, CTS served as an activator of FKBP1A, which led to a reduced activity of the TGFß receptor pathway and therefore to a diminished CK1 and CK10 expression. The combination of the FKBP1A activator CTS with the inhibitor tacrolimus neutralized the effects of both compounds. From these data, a potential dermatological use of CTS and CTS-containing plant extracts (e.g., hydroalcoholic extract from the roots of S. miltiorrhiza) for keratinopathic ichthyosis, a disease characterized by overexpression of CK1 and CK10, is discussed. This study displays an experimental strategy for combining phytochemical aspects on active natural products with systematic identification of molecular targets on gene, protein, and cell level.

ß-1,3/1,4-Glucan Lichenan from Cetraria islandica (L.) ACH. induces cellular differentiation of human keratinocytes.

Lichenan (molecular weight 275 kDa, ß-D-1,3/1,4-glucopyranose ratio 1:3) from Cetraria islandica at a concentration of 100 µg/mL induced terminal cellular differentiation of primary human keratinocytes as demonstrated by immunofluorescence staining using cytokeratin 10 and involucrin as marker proteins. Lichenan-derived oligosaccharides (DP3 to 8), obtained by acid-catalyzed partial hydrolysis of the polymer, did not influence cellular differentiation. Cytokeratin, filaggrin, involucrin, loricrin and transglutaminase gene expression as typical differentiation markers was increased by lichenan in a time-dependent manner. Lichenan upregulated gene cluster which were mostly related to cellular differentiation with focus on MAPK signaling as was shown by Whole Human Genome Microarray. These gene expression data from the array experiments were subsequently confirmed by qPCR for selected genes. For identification of the molecular binding structures of lichenan 1- and 2-D PAGE of keratinocyte protein membrane preparations was performed, followed by blotting with FITC-labeled lichenan and subsequent mass spectrometric identification of the pinpointed proteins. Epidermal growth factor receptor (EGFR) and integrin ß4, both proteins being strongly involved in induction of keratinocyte differentiation were identified. In addition, protein disulfide isomerase A3 (PDIA3) showed strong binding to FITC-lichenan, indicating this enzyme to be an intracellular target of the glucan for induction of the cellular differentiation of keratinocytes. As lichenan did not influence the EGFR phosphorylation and the phosphorylation of CREB transcription factor but strongly interacted with cytosolic proteins it is hypothized that the glucan may interact with EGFR and is subsequently internalized into the cell via endosomal uptake, interacting with PDIA3, which again alters TGFß1 signaling towards keratinocyte differentiation.

Xyloglucan from Tropaeolum majus Seeds Induces Cellular Differentiation of Human Keratinocytes by Inhibition of EGFR Phosphorylation and Decreased Activity of Transcription Factor CREB.

Xyloglucan XG (molecular weight 462 kDa, 1,4-/1,4,6-(pGlc) linked backbone, side chains of 1-pXyl, 1,2-pXyl, 1-p-Gal) was isolated from the seeds of Tropaeolum majus. XG (100 µg/mL) induced terminal cellular differentiation of human keratinocytes, as demonstrated by immunofluorescence staining and Western blot using cytokeratin 10 and involucrin as marker proteins. Differentiation was also induced by XG-derived oligosaccharides (degree of polymerization 7-9). Quantitative real-time polymerase chain reaction (qPCR) revealed the induction of gene expression of typical differentiation markers (cytokeratin, filaggrin, involucrin, loricrin, transglutaminase) in a time-dependent manner. Whole human genome microarray indicated that most of upregulated genes were related to differentiation processes. Microarray findings on selected genes were subsequently confirmed by qPCR. For identification of the molecular target of xyloglucan PAGE of keratinocyte membrane preparations was performed, followed by blotting with fluorescein isothiocyanate-labeled XG. XG interacting proteins were characterized by MS. Peptide fragments of epidermal growth factor receptor (EGFR) and integrin ß4 were identified. Subsequent phospho-kinase array indicated that phosphorylation of EGFR and transcription factor cAMP response element-binding protein (CREB) was decreased in the XG-treated cells. Thus, the XG-induced differentiation of keratinocytes is proposed to be mediated by the inhibition of the phosphorylation of EGFR, leading to a dimished CREB activation, which is essential for the activation of cellular differentiation.