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  • br The quantification of ML and VT


    The quantification of ML and VT contents in the preparation are detailed by Pfüller and Schumacher (2016). Iscucin® Salicis and Tiliae delivered high, Crataegi, Populi and Mali middle and Abietis and Pini low content of ML-1, analysed by ELISA. A high value of VT-A was mea-sured in Iscucin® Crataegi by HPLC-UV.
    2.1.2. Isolated and reference compounds
    Isolation of ML-1 and VT-A is described by Pfüller and Schumacher (2016). Isolated ML-1 (University Hamburg, Hamburg, Germany) and isolated VT-A1 (Abnoba GmbH, Pforzheim, Germany) were used in three concentrations corresponding to the content in the named Iscucin® preparations, 1,000 ng/ml, 10,000 ng/ml and 20,000 ng/ml of ML-1 as well as 10 μg/ml 25 μg/ml and 48.4 μg/ml of VT-A (Table 1). Ad-ditionally nine mixtures of ML-1 and VT-A in varying concentrations, corresponding to the contents in the mistletoe preparations were con-sidered, respectively (Table 1).
    Isotonic solution (WALA Heilmittel GmbH, Bad Boll, Germany) was used to prepare the various concentrations as dilution medium and as negative control (100% cell viability). Staurosporine (10−5 M) (Sigma-Aldrich Chemie GmbH, Munich, Germany) served as positive control (0% cell viability).
    The antineoplastic agent Actinomycin-D (Sigma-Aldrich Chemie GmbH, Munich, Germany) was used as reference substance (Table 1).
    The following tumor cell lines were selected to evaluate the anti-proliferative effect: Caki-2 (renal cell cancer), which is little sensitive to 5-fluorouracil and sorafenib, LN229 (glioblastoma), SK-N-SH (pae-driatic neuroblastoma), HeLa (cervix cancer), HCC827 (non-small cell lung cancer) and DLD-1 (colorectal cancer). 5,000 tumor Fasudil per well were seeded in 150 μl RPMI-1640 supplemented with 10% FCS and Penicillin/Streptomycin (HCC827) or 150 μl DMEM (Caki-1, DLD-1, HeLa, LN229, SK-N-SH) over night at 37 °C.
    The assay was performed by ProQinase GmbH (Freiburg, Germany),
    Table 1
    Description of test items.
    # Test item Company Batch number Characteritics, ingredients, purity
    Viscum album preparation
    VT: middle ML-1 and VT-A from Viscum album
    Ricin RCA120 is highly purified by affinity chromatography using the method of Lin and Li
    Reference thionin
    26 Purothionin: Corystein™, TaKaRa Bio Europe S.A.S. – Molecular weight: 5,000
    Isoelectric point: 10.0
    Purothionin: Corystein™,
    Showed a single band by SDS-polyacrylamide gel electrophoresis.
    Purothionin: Corystein™,
    Controls and reference item
    29 Isotonic solution WALA Heilmittel GmbH A049707B, A056676 Natriumhydrogencarbonate, natriumchloride, water
    30 Staurosporine Sigma-Aldrich Chemie – Purity: > 98% (absorbance at 292 nm); chromatographically homogeneous
    31 Actinomycin-D Sigma-Aldrich Chemie 024M4143V Lyophilized powder with purity of 95% (HPLC).
    modified according to the methods from Page et al. (1993). Test items were added to seeded cells in semi-logarithmic dilution steps (1:100, 1:300, …, 1:100,000, 1:300,000) and Actinomycin-D in a deca-loga-rithmic dilution series (10−6 - 10−13). After 72 h of growth at appro-priate culture conditions, 15 μl resazurin was added and 3 – 5 h later the fluorescence intensity was determined (excitation: 560 nm; emission: 590 nm). Each sample was tested in three independent experiments. Cell viability was expressed in relation to negative and positive control. IC50 calculations were performed using GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, US).
    3. Results and discussion
    3.1. Iscucin® preparations showed antiproliferative effects
    The tumor cell lines analysed reacted differently to the treatment. HCC827 cells (lung cancer) were very sensitive to the Iscucin® 
    treatment. HeLa (cervix cancer) showed a high, Caki-2, SK-N-SH and LN229 a middle and DLD-1 a low sensitivity against all Iscucin® pre-parations. The IC50 concentration of the reference substance Actinomycin-D (0.71 μg/ml) was comparable to Iscucin® Salicis (0.6 μg/ ml), Tiliae (0.6 μg/ml) and Crataegi (0.7 μg/ml), when exposing the lung cancer cell line. Otherwise, Actinomycin-D was more effective (Table 2). Actinomycin-D, which binds to DNA and can inhibit RNA-synthesis and cell proliferation, belongs to the group of cytotoxic an-tibiotics. In general, very strong antiproliferative effects were found from treatment with Iscucin® Salicis and Tiliae and a strong with Iscucin® Crataegi, Mali and Populi. The mean value of the half maximal inhibitory concentration of Iscucin® Mali (48.5 μg/ml) was comparable to results from previous investigations on fermented aqueous mistletoe prepara-tions (Iscador M Spezial: 30.8 μg/ml) (Kelter and Fiebig, 2006). Iscucin® Abietis and Iscucin® Pini showed only weak antiproliferative effects (Table 2). This is in line written previous reports where preparations from conifers (Abietis, Pini) exerted lower antiproliferative action