Design of an Herbal Preparation Composed by a Combination of Ruscus aculeatus L. and Vitis vinifera L. Extracts, Magnolol and Diosmetin to Address Chronic Venous Diseases through an Anti-Inflammatory Effect and AP-1 Modulation

Abstract

Chronic venous disease (CVD) is an often underestimated inflammatory pathological condition that can have a serious impact on quality of life. Many therapies have been proposed to deal with CVD, but unfortunately the symptoms recur with increasing frequency and intensity as soon as treatments are stopped. Previous studies have shown that the common inflammatory transcription factor AP-1 (activator protein-1) and nuclear factor kappa-activated B-cell light chain enhancer (NF-kB) play key roles in the initiation and progression of this vascular dysfunction. The aim of this research was to develop a herbal product that acts simultaneously on different aspects of CVD-related inflammation. Based on the evidence that several natural components of plant origin are used to treat venous insufficiency and that magnolol has been suggested as a putative modulator of AP-1, two herbal preparations based on Ruscus aculeatus root extracts, and Vitis vinifera seed extracts, as well as diosmetin and magnolol, were established. A preliminary MTT-based evaluation of the possible cytotoxic effects of these preparations led to the selection of one of them, named DMRV-2, for further investigation. First, the anti-inflammatory efficacy of DMRV-2 was demonstrated by monitoring its ability to reduce cytokine secretion from endothelial cells subjected to LPS-induced inflammation. Furthermore, using a real-time PCR-based protocol, the effect of DMRV-2 on AP-1 expression and activity was also evaluated; the results obtained demonstrated that the incubation of the endothelial cells with this preparation almost completely nullified the effects exerted by the treatment with LPS on AP-1. Similar results were also obtained for NF-kB, whose activation was evaluated by monitoring its distribution between the cytosol and the nucleus of endothelial cells after the different treatments.

Keywords: AP-1; MCP-1; Ruscus aculeatus L.; Vitis vinifera L.; anti-inflammatory effects; diosmetin; magnolol; transcriptional factors; venous disease.

Figure 1

Schematic representation of chronic venous disease (CVD) pathogenesis and major clinical signs.

Figure 2

HUVECs viability measured by MTT assay (Mean ± SEM of 6 independent measurements) following different treatments with each compound and extract selected: the viability of HUVEC cells after a 24 h incubation with different concentrations of magnolol (MGL) (1.3, 2.5, 5.0, 10.0 μg/mL) (A); diosmetin (DSM) (3.1, 6.3, 12.5, 25.0 μg/mL) (B); R. aculeatus root extract (RSC) (6.3, 12.5, 25.0, 50.0 μg/mL) (C); and V. vinifera seeds extract (V.Vin) (6.3, 12.5, 25.0, 50.0 μg/mL) (D). In all the panels, CTRL- represents the negative control, consisting of HUVECs incubated with the cell medium for 24 h. * p < 0.05 vs. CTRL-.

Figure 3

HUVECs viability measured by MTT assay (Mean ± SEM of 6 independent measurements) following different treatments with the two herbal preparations. The cell viability percentage of HUVECs measured after 24 h of incubation with two different concentrations (25 mg/mL and 40 mg/mL) of DMRV-1 (diosmetin 6%, magnolol 10%, V. vinifera seeds extract 48%, R. aculeatus root extract 36%) and DMRV-2 (diosmetin 11%, magnolol 9%, V. vinifera seeds extract 45%, R. aculeatus root extract 35%) is shown. CTRL-represents the negative control, consisting of HUVECs incubated with the cell medium for 24 h. * p < 0.05 vs. CTRL-.

Figure 4

Analysis of IL-6 and IL-8 secretion in HUVECs. HUVECs were stimulated with 0.1 µg/mL LPS for 4 h and then incubated with DMVR-2 or underwent change of the medium. ELISA were performed to measure the concentration of IL-6 and IL-8 released after LPS stimulation, and after 3 h or 6 h of the different incubations. * p < 0.05 vs. respective controls.

Figure 5

NF-κB translocation analysis. HUVECs were stimulated with 0.1 µg/mL LPS for 4 h and then incubated with DMVR-2 or underwent change of the medium. A WB-based measurement of the NF-κB concentration in the HUVEC cytosol or nucleus following the different treatments was performed. The results of densitometric analysis of the resulting bands, normalized towards the suitable control (nucleolin for nucleus and GAPDH for cytosol) are reported. * p < 0.05 vs. respective controls.

Figure 6

RT-qPCR-measured levels of MCP-1 (A), c-Jun (B) and c-Fos (C) mRNAs in HUVECs following different treatments. HUVECs were stimulated with 0.1 µg/mL LPS for 4 h and then incubated with DMVR-2 or underwent change of the medium. RT-qPCR analyses were performed after LPS stimulation, and after 3 h or 6 h of different incubations. * p < 0.05 vs. respective controls.