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  • Journal of Ethnopharmacology br reaction

    2022-05-22

     Journal of Ethnopharmacology 236 (2019) 100–107
    reaction of the fluorescent cell viability dye. Living GW9662 reduce blue resazurin into the pink colored and highly red fluorescent resorufin (Präbst et al., 2000).
    2. Material and methods
    2.1.1. Aqueous mistletoe preparations
    Iscucin® (WALA Heilmittel GmbH, Bad Boll, Germany) is an aqueous preparation from mistletoe (Viscum album L.). The mistletoes were collected from seven host trees: fir tree (Abietis), hawthorn (Crataegi), apple tree (Mali), pine tree (Pini), poplar tree (Populi), willow tree (Salicis) and lime tree (Tiliae). Iscucin® using the fresh plant was carried out according to the official production protocol described in the German Homoeopathic Pharmacopoeia (GHP) version 38. The fresh mistletoes were cultivated on and harvested from WALA Heilmittel GmbH dedicated cultivation areas (Zuzak et al., 2004). Identification and harvest of plant material was carried out by trained staff according to the principles of GACP (good agricultural collection practice), i. e. according to valid requirements for the manufacturing of medicinal products in Europe. In this study strength H was used. This means a concentration of 5% (1:20) plant extract in purified water.
    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 cells 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