Stability of protoanemonin in plant extracts from Helleborus niger L. and Pulsatilla vulgaris Mill.

The concentration levels and stability of protoanemonin, a characteristic constituent of Ranunculaceae species with antimicrobial and fungicidal properties, were studied for the first time in plant extracts prepared from Helleborus niger L. and Pulsatilla vulgaris Mill. using fermentative production processes. Protoanemonin levels quantified by HPLC-DAD analysis were 0.0345 and 0.0662 mg/g in two freshly prepared Helleborus (whole, flowering plant) extracts and 0.3875 mg/g and 0.4193 mg/g in Pulsatilla (flowers) extracts. Protoanemonin proved to be rather instable in aqueous-fermented extracts stored at 15 °C in the dark, and its concentration decreased rapidly over 12 months of storage independently of the plant species. The decrease was most pronounced when initial concentrations were high (decrease by about 70%). In contrast, low protoanemonin levels remained stable in solution for more than 12 months. Anemonin, the dimer of protoanemonin, was detected in increasing concentrations only in Pulsatilla samples, but its concentration only accounted for less than 50% of the theoretically expected amount. With respect to fermented extracts, both physical processes such as self-polymerization and adsorption/binding to other extract constituents as well as biodegradation were concluded to be responsible for protoanemonin decline. As opposed to plant extracts, both protoanemonin and anemonin levels decreased in 0.22 µm-filtered samples stored in vials. This may be explained by a reduced release from plant material in combination with physicochemically induced degradation. Reduction was most pronounced upon light exposure and elevated temperatures, clearly indicating that photochemical degradation is involved. Contents of protoanemonin in a set of extract batches were 0.0896 ± 0.0125 mg/g and 0.0618 ± 0.0180 mg/g in Helleborus and Pulsatilla extracts, and anemonin levels were 0.0230 ± 0.0076 mg/g and 0.0482 ± 0.0282 mg/g, respectively. Due to its antibiotic effects, but also its reactivity, protoanemonin is a therapeutically and toxicologically relevant constituent, and its concentration should therefore be carefully monitored.

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.

Differential cytotoxic properties of Helleborus niger L. on tumour and immunocompetent cells.

ETHNOPHARMACOLOGICAL RELEVANCE: In Romanian folk medicine, Helleborus niger L. is used for the treatment of rheumatoid arthritis or viral infections and in complementary therapy, especially in anthroposophic medicine (AM), where the plant is administered as an adjuvant to treat malignant diseases. In the present study, we investigated the differential cytotoxic effects of H. niger on human tumour and healthy cells of the human immune system in vitro. MATERIAL AND METHODS: Protoanemonin and saponins, as significant constituents of H. niger extracts, were quantified in five individual batches using validated HPLC methods. Further, the impact of H. niger on proliferation capacity (MTT assay) as well as on apoptosis and necrosis induction in a panel of tumour cell lines and human lymphocytes (combined annexin V and propidium iodide staining) was monitored. In addition, NK cell function (degranulation-CD107a assay and IFN-gamma secretion) was also investigated since these immunocompetent cells are important for the control of malignancies within the human body. RESULTS: Extracts of H. niger induced proliferation inhibition not only of lymphoblastic leukaemia cells (MOLT4; IC50: 171 µg/mL) but also of myosarcoma (SK-UT-1b; IC50: 304 µg/mL) and melanoma cells (HT-144; IC50: 569 µg/mL) due to the induction of apoptosis. Purified T cells or NK cells were significantly affected through the presence of high H. niger concentrations while bulk lymphocytes were not affected. NK cells' anti-tumour functions expressed by degranulation capacity as well as IFN-y production were unaffected by the presence of the H. niger extract. Since protoanemonin and saponins have been reported in the literature to exert cytotoxic effects, their content was also determined. CONCLUSIONS: H. niger extracts exhibit differential cytotoxicity towards tumour cell lines and healthy human T- and NK-cells.

Reconsidering the quantitative analysis of organic carbon concentrations in size exclusion chromatography.

The evaluation of the molecular size distribution of natural organic matter (NOM) in aquatic environments via size exclusion chromatography (SEC) is important for the understanding of environmental processes such as nutrient cycling and pollutant transport as well as of technical water treatment processes. The use of organic carbon (OC) detectors has become popular in recent studies due to improved availability and quantification possibilities, which supposedly are superior to those of ultraviolet (UV) detectors. A set of 12 NOM samples was used to demonstrate the limitations of online OC detection (OCD) when analyzing complex aquatic organic matter. A novel evaluation approach for SEC data is introduced by combining the information from UV absorbance (UVA) and OCD chromatograms as well as offline total OC (t-OC) and dissolved OC-specific UVA (SUVA) measurements. It could be shown that about 70% of certain OC components were not detected with the OCD system used in this study. For the investigated samples, these types of carbon accounted for up to 72% of the t-OC, i.e. for such NOM samples quantification by OCD is not possible or at least highly questionable. The addition of an oxidant improved the overall oxidation efficiency only slightly. Most likely NOM that predominantly consists of polysaccharides and features a nominal molecular weight of 150kg/mol or more was responsible for low OCD yields. For future applications, a further improvement of the OCD system would be worthwhile so that quantitative analytical data on the molecular size distribution of NOM and its structural characteristics such as the SUVA distribution can be obtained.

Ultrafiltration of nonionic surfactants and dissolved organic matter.

A brownwater sample with a high content of humic substances (HS) was fractionated by multistage ultrafiltration (mst-UF) into five fractions with nominal molecular weights ranging from >30 to <1 kDa. Fractions were characterized with respect to molecular size distribution and structure. Size exclusion chromatography with online DOC detection revealed that mst-UF yielded fractions with decreasing Mp (molecular weight at peak maximum) and polydispersities from nominally large to small mst-UF fractions. 13C MAS NMR analysis showed that the content of carbohydrate structures decreased from the original sample toward smaller molecular weight (MW) fractions, which in turn contained more carboxylic groups and branched aliphatic structures. Specific UV absorbances (SUVA254) were highest in the >30 kDa fraction and decreased with decreasing MW. To evaluate whether separation mechanisms other than size exclusion were of importance during the fractionation, the behavior of low molecular weight model compounds (MC) with a range of polarities was studied. Recoveries decreased with increasing hydrophobicity of the MC. For selected nonylphenol ethoxylates and 4-nonylphenol the recovery correlated well with the hydrophile-lipophile balance value. The presence of dissolved organic matter (DOM) caused an additional loss of hydrophobic MC, possibly because of sorption of the compounds onto DOM fouling layers. The hydrophilic MC caffeine was recovered almost completely (85-86%) regardless of the DOM content of the model solution. It was concluded that size exclusion was the dominant fractionation mechanism for caffeine, whereas hydrophobic interactions played a major role during the mst-UF fractionation of nonpolar contaminants. For a better understanding of the behavior of polyfunctional molecules such as HS, the effect of other physicochemical properties needs to be investigated in further studies.

A new concept for the fractionation of DOM as a basis for its combined chemical and biological characterization.

To overcome the problem of non-specific interactions during the preparative fractionation of dissolved organic matter (DOM) for its combined chemical and biological characterization, the concept of standardized fractions (SFs) was developed. The concept is based on the calibration of the fractionation systems with suitable standards for the experimental definition of SFs, and the subsequent preparative fractionation of DOM samples according to the predefined SFs. Undesired non-specific interactions can thus be minimized experimentally. It was applied to the fractionation methods size exclusion chromatography (SEC) and multistage ultrafiltration (mst-UF). A brownwater sample and an effluent from a sewage treatment plant were fractionated. The two methods performed rather differently which was evidenced both by the polydispersity of the resulting fractions as well as sum parameter fractionation patterns. Consequently, the interpretation of data obtained from the chemical-physical as well as biological analysis must always consider the limitations and particularities of the fractionation method employed.