Guido Jürgenliemk

Phloroglucinol derivatives from Hypericum empetrifolium with antiproliferative activity on endothelial cells

Five acylphloroglucinols substituted with monoterpenoids (empetrifelixin A-D and empetrikajaforin), three known monocyclic acylphloroglucinols and one monocyclic acylphloroglucinol were isolated from a petrol ether extract of Hypericum empetrifolium after fractionation by flash chromatography on silica gel, RP-18 and subsequent purification by preparative HPLC (RP-18). Their structures were elucidated by 1D, 2D NMR techniques and HREIMS. To determine a possible anti-angiogenic activity, inhibition of cell proliferation was measured using a human microvascular endothelial cell line (HMEC-1). Subconfluent grown HMEC-1 cells were treated with all compounds isolated in sufficient amounts and stained with crystal violet. Highest activity was observed for empetrifelixin A and empetrifelixin D showing a concentration dependent inhibition of cell proliferation with IC(50) values of 6.5 ± 0.1 and 7.3 ± 0.4 μM, respectively. Empetrifelixin A also showed activity in a cell migration assay with HMEC-1 cells in low micromolar concentrations.

A novel approach to prevent endothelial hyperpermeability: The Crataegus extract WS® 1442 targets the cAMP/Rap1 pathway

Endothelial hyperpermeability followed by edema formation is a hallmark of many severe disorders. Effective drugs directly targeting endothelial barrier function are widely lacking. We hypothesized that the hawthorn (Crataegus spp.) extract WS® 1442, a proven multi-component drug against moderate forms of heart failure, would prevent vascular leakage by affecting endothelial barrier-regulating systems. In vivo, WS® 1442 inhibited the histamine-evoked extravasation of FITC-dextran from mouse cremaster muscle venules. In cultured human endothelial cells, WS® 1442 blocked the thrombin-induced FITC-dextran permeability. By applying biochemical and microscopic techniques, we revealed that WS® 1442 abrogates detrimental effects of thrombin on adherens junctions (vascular endothelial-cadherin), the F-actin cytoskeleton, and the contractile apparatus (myosin light chain). Mechanistically, WS® 1442 inhibited the thrombin-induced rise of intracellular calcium (ratiometric measurement), followed by an inactivation of PKC and RhoA (pulldown assay). Moreover, WS® 1442 increased endothelial cAMP levels (ELISA), which consequently activated PKA and Rap1 (pulldown assay). Utilizing pharmacological inhibitors or siRNA, we found that PKA is not involved in barrier protection, whereas Epac1, Rap1, and Rac1 play a crucial role in the WS® 1442-induced activation of cortactin, which triggers a strong cortical actin rearrangement. In summary, WS® 1442 effectively protects against endothelial barrier dysfunction in vitro and in vivo. It specifically interacts with endothelial permeability-regulating systems by blocking the Ca(2+)/PKC/RhoA and activating the cAMP/Epac1/Rap1 pathway. As a proven safe herbal drug, WS® 1442 opens a novel pharmacological approach to treat hyperpermeability-associated diseases. This in-depth mechanistic work contributes to a better acceptance of this herbal remedy.

Bi-, tri-, and polycyclic acylphloroglucinols from Hypericum empetrifolium.

The ¹H NMR-guided fractionation of a petroleum ether extract of Hypericum empetrifolium led to the isolation of four new bicyclic (1-4), four known bicyclic (5-8), three new tricyclic (9-11), and three new polycyclic acylphloroglucinols (12/13 and 14) possessing a monoterpenoid citran moiety. Compounds 12/13 were isolated as a mixture of two inseparable structural isomers. The compounds showed in vitro antiproliferative activity against human microvascular endothelial cells (HMEC-1) with IC₅₀ values in the range 9.2 ± 2.0 to 29.6 ± 3.5 μM.

Catechol, a Bioactive Degradation Product of Salicortin, Reduces TNF-α Induced ICAM-1 Expression in Human Endothelial Cells

The phenolic glucoside salicortin was isolated from a Willow bark extract, and its ability to reduce the TNF- α induced ICAM-1 expression (10 ng/mL, 30 min pretreatment with salicortin) was tested IN VITRO on human microvascular endothelial cells (HMEC-1). After 24 h, 25 µM salicortin decreased the TNF- α induced ICAM-1 expression to 65.9 % compared to cells which were treated only with TNF- α. In parallel, the stability of 25 µM salicortin under assay conditions was determined by HPLC. Within 24 h, the salicortin concentration decreased to 3.1 µM whereas catechol, a known NF- κB inhibitor, rose as a metabolite. After 8 h the catechol concentration was relatively constant and varied between 8.2 and 10.9 µM. Considering this degradation in the IN VITRO test system, 10 µM catechol was added 8 h after TNF- α stimulation, and 16 h later the ICAM-1 expression was determined. In this setting, the ICAM-1 expression was reduced to 74.8 %. This is comparable to the effect obtained from 25 µM salicortin and indicates that its activity is related to the generation of catechol, as salicin, saligenin, and salicylic acid are only marginally active or inactive in this test system in a concentration up to 50 µM. These results indicate catechol as an important bioactive metabolite from salicortin.

Novel Phenyl-1-benzoxepinols from Butcher's Broom (Rusci rhizoma)

Two novel compounds, (3S)‐2,3‐dihydro‐3‐(4‐hydroxyphenyl)‐1‐benzoxepin‐8‐ol (1; ruscozepine A) and (3S)‐2,3‐dihydro‐3‐(4‐hydroxyphenyl)‐8‐methoxy‐1‐benzoxepin‐7‐ol (2; ruscozepine B) were isolated from butchers broom (Rusci rhizoma) together with a biosynthetically possible phenylethanoid precursor, hydroxytyrosol (3). The structures were elucidated by spectroscopic methods such as 1D‐ and 2D‐NMR (COSY, HSQC, HMBC, ROESY), and HR‐EI‐MS experiments. The absolute configuration of the ruscozepines was determined by electronic circular dichroism.

Bioactive cycloartane-type triterpene glycosides from Astragalus elongatus.

Abstract Together with two known cycloartane-type glycosides, askendosides D (3-O-[α-arabinopyranosyl-( 1→2)-β-xylopyranosyl]-6-O-β-xylopyranosyl-cycloastragenol, 2) and G (3-O- [α-arabinopyranosyl-(1→2)-β-xylopyranosyl]-16-O-β-glucopyranosyl-3β,6α,16β,24(R),25- pentahydroxycycloartane, 3), also a new monodesmosidic cycloartane-type glycoside, elongatoside (1), was isolated from the roots of Astragalus elongatus and identified as 3-O- [α-arabinopyranosyl-(1→2)-β-xylopyranosyl]-cycloastragenol. All structures were unambiguously determined by means of spectroscopic and spectrometric methods (1D and 2D NMR, ESI-MS). The isolated compounds were tested for the inhibition of proliferation and ICAM-1 expression in vitro using the human microvascular endothelial cell line (HMEC-1). 1 showed weak activity in the ICAM-1 assay.

Revisited anti-inflammatory activity of matricine in vitro: Comparison with chamazulene

The proazulene matricine (1) is present in chamomile flower heads and has been proven to exhibit strong in vivo anti-inflammatory activity. In contrast to other secondary metabolites in chamomile preparations like its degradation product chamazulene (2), no plausible targets have been found to explain this activity. Therefore we revisited 1 regarding its in vitro anti-inflammatory activity in cellular and molecular studies. Using ICAM-1 as a marker for NF-κB activation, it was shown that ICAM-1 protein expression induced by TNF-α and LPS, but not by IFN-γ, was remarkably inhibited by 1 in endothelial cells (HMEC-1). Inhibition was concentration-dependent in a micromolar range (10-75 μM) and did not involve cytotoxic effects. At 75 μM expression of the adhesion molecule ICAM-1 was down to 52.7 ± 3.3% and 20.4 ± 1.8% of control in TNF-α and LPS-stimulated HMEC-1, respectively. In contrast, 2 showed no activity. Quantitative RT-PCR experiments revealed that TNF-α-induced expression of the ICAM-1 gene was also reduced by 1 in a concentration-dependent manner, reaching 32.3 ± 6.2% of control at 100 μM matricine. Additional functional assays (NF-κB promotor activity and cytoplasm to nucleus translocation) confirmed the inhibitory effect of 1 on NF-κB signaling. Despite the fact that 1 lacks an α,β-unsaturated carbonyl and is thus not able to act via a Michael reaction with electron rich SH groups of functional biological molecules, data gave strong evidence that 1 inhibits NF-κB transcriptional activity in endothelial cells by an hitherto unknown mechanism and this may contribute to its well-known anti-inflammatory activity in vivo.

Salix daphnoides: A Screening for Oligomeric and Polymeric Proanthocyanidins

In the present study, a qualitative analysis of proanthocyanidins (PAs) from an aqueous-methanolic extract of Salix daphnoides VILL. bark is described. Procyanidin B1 (1), B2 (2), B3 (3), B4 (4), C1 (5), epicatechin-(4β→8)-epicatechin-(4β→8)-catechin (6) and epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin-(4β→8)-catechin (7) have been isolated by a combination of different chromatographic separations on Sephadex® LH-20-, MCI®-, Diol-and RP-18-phases. Mass spectrometry, 1D- and 2D-NMR, circular dichroism and polarimetry were used for their structure elucidation and verification by comparison with the literature. Additionally, two fractions of very polar flavan-3-ols were compared: “regular” polymeric PAs received at the very end of the Sephadex® LH-20 chromatography showing no mobility on silica TLC and “unusual” PAs with the same RF-value but already eluting together with flavonoids in the Sephadex® LH-20 system. These “unusual” PAs were subsequently enriched by centrifugal partition chromatography (CPC). 13C-NMR, polarimetry, thiolysis, acid hydrolysis and phloroglucinol degradation were used to characterize both fractions. Differences in the composition of different flavan-3-ol units and the middle chain length were observed.

Catechol conjugates are in vivo metabolites of Salicis cortex.

After oral administration of 100 mg/kg b. w. (235.8 µmol/kg) salicortin to Wistar rats, peak serum concentrations of 1.43 mg/L (13.0 µM) catechol were detected after 0.5 h in addition to salicylic acid by HPLC-DAD after serum processing with β-glucuronidase and sulphatase. Both metabolites could also be detected in the serum of healthy volunteers following oral administration of a willow bark extract (Salicis cortex, Salix spec., Salicaceae) corresponding to 240 mg of salicin after processing with both enzymes. In humans, the cmax (1.46 mg/L, 13.3 µM) of catechol was reached after 1.2 h. The predominant phase-II metabolite in humans and rats was catechol sulphate, determined by HPLC analysis of serum samples processed with only one kind of enzyme. Without serum processing with glucuronidase and sulphatase, no unconjugated catechol could be detected in human and animal serum samples. As catechol is described as an anti-inflammatory compound, these results may contribute to the elucidation of the mechanism of the action of willow bark extract.

Catechol is a bioactive metabolite of Willow bark

It has recently been shown in a bioassay guided fractionation of a Willow bark extract (Salicis cortex, Salix spec., Salicaceae) that not the isolated compound salicortin itself but its degradation product catechol was responsible for an anti-inflammatory effect in vitro. To investigate if this in vitro effect has impact on the in vivo situation, a pharmacokinetic study was performed in humans with eight healthy volunteers. After oral administration of a Willow bark extract corresponding to 240 mg salicin, venous blood samples were taken up to 24 hours, and serum was analyzed by HPLC-DAD after processing with glucuronidase and sulfatase. Mean peak concentrations of 13.3µM catechol were observed after 1.2 hours. No free catechol could be detected without enzyme processing and the predominant phase-II-metabolite was catechol sulfate. To exclude that the detected catechol in the human serum samples was not solely absorbed as a genuine compound of the Willow bark extract, pure salicortin 100 mg/kg b.w. was orally administered to Wistar rats. In this study, catechol could definitely be proved as a metabolite of salicortin in serum with a cmax of 13.0µM after 0.5h. These findings indicate that not salicortin per se but rather catechol, which is one of its active metabolites reported in the literature to have anti-inflammatory properties, contributes towards the overall ant-inflammatory and analgesic efficacy of Willow bark.