Biochemometry identifies suppressors of pro-inflammatory gene expression in Pterocarpus santalinus heartwood.

The heartwood extract of the Ayurvedic medicinal plant Pterocarpus santalinus L. f. has previously been shown to significantly suppress the expression of CX3CL1 and other pro-inflammatory molecules in IL-1-stimulated human endothelial cells. Here, we identify the pigment-depleted extract PSD as the most promising yet still complex source of metabolites acting as an inhibitor of CX3CL1 gene expression. For the target-oriented identification of the constituents contributing to the observed in vitro anti-inflammatory effect of PSD, the biochemometric approach ELINA (Eliciting Nature's Activities) was applied. ELINA relies on the deconvolution of complex mixtures by generating microfractions with quantitative variances of constituents over several consecutive fractions. Therefore, PSD was separated into 35 microfractions by means of flash chromatography. Their 1H NMR data and bioactivity data were correlated by heterocovariance analysis. Complemented by LC-MS-ELSD data, ELINA differentiated between constituents with positive and detrimental effects towards activity and allowed for the prioritization of compounds to be isolated in the early steps of phytochemical investigation. A hyphenated high-performance counter-current chromatographic device (HPCCC+) was employed for efficient and targeted isolation of bioactive constituents. A total of 15 metabolites were isolated, including four previously unreported constituents and nine that have never been described before from red sandalwood. Nine isolates were probed for their inhibitory effects on CX3CL1 gene expression, of which four isoflavonoids, namely pterosonin A (1), santal (6), 7,3'-dimethylorobol (12) and the previously unreported compound pterosantalin A (2), were identified as pronounced inhibitors of CX3CL1 gene expression in vitro.

Pterocarpus santalinus Selectively Inhibits a Subset of Pro-Inflammatory Genes in Interleukin-1 Stimulated Endothelial Cells.

Based on the traditional use and scientific reports on the anti-inflammatory potential of red sandalwood, i.e., the heartwood of Pterocarpus santalinus L., we investigated its activity in a model of IL-1 stimulated endothelial cells. Endothelial cells were stimulated with IL-1 with or without prior incubation with a defined sandalwoodextract (PS), and analyzed for the expression of selected pro-inflammatory genes. The activity of NF-κB, a transcription factor of central importance for inflammatory gene expression was assessed by reporter gene analysis, Western blotting of IκBα, and nuclear translocation studies. In addition, microarray studies were performed followed by verification of selected genes by qPCR and supplemented by bioinformatics analysis. Our results show that PS is able to suppress the induction of E-selectin and VCAM-1, molecules that mediate key steps in the adhesion of leukocytes to the endothelium. It also suppressed the activity of an NF-κB reporter, IκBα phosphorylation and degradation, and the nuclear translocation of NF-κB RelA. In contrast, it stimulated JNK phosphorylation indicating the activation of the JNK signaling pathway. Gene expression profiling revealed that PS inhibits only a specific subset of IL-1 induced genes, while others remain unaffected. Most strongly suppressed genes were the signal transducer TRAF1 and the chemokine CX3CL1, whereas IL-8 was an example of a non-affected gene. Notably, PS also stimulated the expression of certain genes, including ones with negative regulatory function, e.g., members of the NR4A family, the mRNA destabilizing protein TTP as well as the transcription factors ATF3 and BHLHB40. These results provide mechanistic insight into the anti-inflammatory activity of PS, and suggest that it acts through the interplay of negative and positive regulators to achieve a differential inhibition of inflammatory gene expression.

Insulin Mimetic Properties of Extracts Prepared from Bellis perennis.

Diabetes mellitus (DM) and consequential cardiovascular diseases lead to millions of deaths worldwide each year; 90% of all people suffering from DM are classified as Type 2 DM (T2DM) patients. T2DM is linked to insulin resistance and a loss of insulin sensitivity. It leads to a reduced uptake of glucose mediated by glucose transporter 4 (GLUT4) in muscle and adipose tissue, and finally hyperglycemia. Using a fluorescence microscopy-based screening assay we searched for herbal extracts that induce GLUT4 translocation in the absence of insulin, and confirmed their activity in chick embryos. We found that extracts prepared from Bellis perennis (common daisy) are efficient inducers of GLUT4 translocation in the applied in vitro cell system. In addition, these extracts also led to reduced blood glucose levels in chicken embryos (in ovo), confirming their activity in a living organism. Using high-performance liquid chromtaography (HPLC) analysis, we identified and quantified numerous polyphenolic compounds including apigenin glycosides, quercitrin and chlorogenic acid, which potentially contribute to the induction of GLUT4 translocation. In conclusion, Bellis perennis extracts reduce blood glucose levels and are therefore suitable candidates for application in food supplements for the prevention and accompanying therapy of T2DM.