br Corresponding author E mail addresses jennifer felenda wa
∗ Corresponding author. E-mail addresses: [email protected] (J.E. Felenda), [email protected] (F.C. Stintzing).
Belonging to the group of thionins, small, cysteine-rich polypeptides (Bohlmann and Apel, 1991), several VTs occur in mistletoe, which have been designated A1, A2, A3, B, B2, C1 and 1-PS (Kahle et al., 2005; Romagnoli et al., 2003; Schaller et al., 1998). They are also present in grains, oats, rice, barley and wheat (Stec, 2006). The thionins crambin, β-hordothionin and α-purothionin are structurally similar to VT (Bohlmann and Apel, 1991; Florack and Stiekema, 1994; Orrù et al., 1997; Stec, 2006). All thionins except crambin are cytotoxic. Some have antimicrobial and fungicide properties and others interact with the RNA or DNA of the cells (Bohlmann and Apel, 1991; Florack and Stiekema, 1994; Stec, 2006). VT has been assigned cytotoxic activity against dif-ferent types of tumor cells (Urech and Ramm, 1997; Urech et al., 1995) and strong immunomodulatory eﬀects (Stec, 2006). Purothionin, pur-ified from the endosperm of wheat, has antimicrobial properties, in-hibits protein kinase C and binds to calmodulin (Stec, 2006).
Beside mistletoe, lectins are also detected in many other plants such as banana, wheat, corn, beans and peanuts (Kreis, 2016). The first isolated lectin was the highly toxic ricin, produced in the seeds of the castor oil plant (Ricinus communis L.). From Viscum album L. three lec-tins, which diﬀer in their molecular weight, are known as ML-1, ML-2 and ML-3, respectively (Luther and Becker, 1987; Pfüller, 2000). ML-1 is structurally very similar to ricin (Dietrich et al., 1992; Krauspenhaar et al., 1999; Stirpe et al., 1992). ML-1 is a heterodimeric glycoprotein that has two polypeptide chains linked by a disulphide bond. The A chain possesses RNA N-glycosidase activity while the B chain shows carbohydrate binding properties (Franz, 1986 a; Soler et al., 1996, 1998). The B chain is equipped with a galactose-specific binding do-main (Franz, 1986 a; Stirpe et al., 1992; Soler et al., 1998). Lectins agglutinate erythrocytes or aggregate cells and inhibit protein synth-esis. They also induce apoptosis and Cyclosporin H arrest and thus inhibit angiogenesis and cell proliferation (Beztsinna et al., 2018; Ferriz-Martinez et al., 2010; González De Mejía and Prisecaru, 2007; Kreis, 2016). Moreover lectins aﬀect the immune system by activating the immune cells (González De Mejía and Prisecaru, 2007).
Mistletoes were used in herbal remedies since ancient time. First mistletoe was used against arteriosclerosis, hypertension and other cardiovascular diseases (HMPC, 2012) and in 1917 mistletoe prepara-tions were introduced in cancer therapy. Up till today, cancer patients often use complementary medicine (Molassiotis et al., 2005), especially mistletoe preparations (Van der Weg and Streuli, 2003; Weis et al., 1998).
An evaluation of the pharmacological potential of both isolated substances and the aqueous mistletoe extract (Iscucin®) appeared to be of interest. A causal relationship between compound quantity and pharmacological eﬀect of multicompound blends cannot be drawn ea-sily. Therefore by comparison of the whole plant extract with isolated compounds, specific eﬀects on living cell systems were investigated. The results obtained from these preclinical investigations shall help to better understand the actions of Viscum album on healthy and patho-logical cell systems and shall deepen our understanding into the ther-apeutic potential of mistletoe preparations, especially for the treatment of oncologic events. 2D Alamar Blue Assay is widely used and accepted in oncology for analysis of the impact of anticancer drugs. It is based on the quantification of the population of living cells with the redox Journal of Ethnopharmacology 236 (2019) 100–107
reaction of the fluorescent cell viability dye. Living cells 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.