Preclinical evaluation of safety and potential of black hellebore extracts for cancer treatment.

BACKGROUND: The therapeutic use of Helleborus niger L. is manifold due to its specific phytochemical composition. Two compound groups, the ranunculin derivates including protoanemonin and the steroidal saponins, are also associated with toxicity (genotoxicity, disintegration of membrane structures). Therefore, in vitro investigations were performed on safety aspects of a Helleborus niger aqueous fermented extract (HNE). In addition its therapeutic potential against various cancer cell lines was assessed to gain insight into the respective mechanisms of action. METHODS: To evaluate the safe use of HNE, Ames and hemolytic tests were carried out. Two angiogenesis assays in 2D and 3D design were conducted to assess the anti-angiogenetic potential, for which human umbilical vein endothelial cells (HUVEC) were chosen. A panel of tumor cell lines was used in 2D and 3D proliferation assays as well in the migration- and invasion-assay. All investigations were performed with HNE compared to reference substances. The 2D proliferation assay was additionally performed with isolated compounds of HNE (characteristic steroidal saponins). RESULTS: HNE did not exhibit any genotoxic potential. Concentrations up to 10 µl/ml were classified as non-hemolytic. HNE exerted anti-angiogenetic effects in HUVEC and anti-proliferative effects in five cancer cell lines, whereas hellebosaponin A and D as well macranthosid I did not show comparable effects neither singly nor in combination. Due to the inherent instability of protoanemonin in isolated form, parallel investigations with protoanemonin could not be performed. HNE (600-1000 µg/ml) inhibited the migration of certain cancer cells by > 80% such as Caki-2, DLD-1 and SK-N-SH. CONCLUSION: HNE exhibit neither genotoxic nor hemolytic potential. The present investigations verify the anti-angiogenetic effects on HUVEC, the anti-proliferative effects and migration-inhibiting properties on tumor cells. The lower effect of the relevant steroidal saponins compared to the whole extract underlines the fact that the latter is more effective than a blend of isolated pharmacologically active components.

Constituents from oak bark (Quercus robur L.) inhibit degranulation and allergic mediator release from basophils and mast cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Oak bark has been used since ancient times in Europaen ethnomedicine because of its adstringent, antimicrobial and hemostatic features, e.g. as a remedy for the treatment of wounds and skin diseases. PURPOSE: Oak bark tannins are considered as bioactive natural products, interacting with surface proteins of mucous membranes and might be beneficial for the treatment of allergic diseases. This study investigated the effect of an oak bark decoction (OBD) and isolated tannin fractions on the degranulation capacity and cytokine/chemokine release from rat basophilic cells and human mast cells in vitro, which are essential for the initiation of early- and late-phase allergic reactions. METHODS AND METHODS: By chromatographic separation on Sephadex® LH-20 high- and low-molecular weight tannins were separated from OBD and the tannin composition analyzed by HPLC(DAD)-MSn. Then, the OBD and its fractions were tested in degranulation (ß-hexosaminidase activity) of allergen-specific-activated basophilic cells in a photometric assay. RESULTS: The OBD and the high-molecular tannin fraction showed a dose-dependent inhibition of cell degranulation. Furthermore, the OBD and particularly its high molecular weight tannin fraction exhibited an inhibitory activity on the IL-8-, IL-6- and TNF-α-secretion from stimulated human mast cells, detected and quantified by ELISA. CONCLUSION: The OBD and its high-molecular weight tannins revealed an impact on allergic mediator release of basophilic cells and human mast cells and thereby provide a rationale for the topical treatment with OBD preparations.