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Polysaccharides from fresh Viscum album L. berry extract and their interaction with Viscum album agglutinin I.

A special microfiltrated colloidal preparation from fresh Viscum album L. berries was investigated concerning the occurrence and structural features of polymeric carbohydrates. A crude polysaccharide fraction was isolated from lectin-, tannin- and protein-depleted microfiltrates. Further fractionation by exchange chromatography revealed a neutral fraction (average molecular weight 30 kDa) and three acidic fractions (average molecular weights 1300 kDa). Structural analysis of the respective polysaccharide fractions by quantitative and qualitative determination of the sugar composition and linkage analysis indicated that all acidic fractions contained an acidic arabinogalactan with a rhamnose-galactoronic acid backbone and highly branched arabinose-galactose side chains attached by the rhamnose residues to the backbone. The neutral fraction consisted of a neutral arabinogalactan beside minor amounts (about 10%) of a low substituted xyloglucan. Further studies on interaction between the 1340 kDa acidic rhamno-arabinogalactans II and III and mistletoe lectin Viscum album agglutinin I (VAA I) revealed binding capacities between these compounds, while the neutral polymers interacted significantly less with VAA I. Only partial binding of VAA I was observed by incubation of the lectin with polysaccharide II. Similar interactions of polysaccharide fraction III with VAA I was measured in a BIACRORE biosensore system. Using the hemagglutination test, increased agglutination of erythrocytes was observed when mistletoes lectin I and the respective polysaccharide fractions were present together in the assay. All these data indicate clearly strong interaction between VAA I and Viscum polysaccharides.

Adult↗

Comparative study of Korean (Viscum album var. coloratum) and European mistletoes (Viscum album).

A lectin (agglutinin, VCA) from Korean mistletoe (Viscum album L. coloratum) was isolated by affinity chromatograpy on a asialofetuin-Sepharose 4B. The molecular weights of A- and B-chains of VCA were differenf from those of VAAS. The VCA recognized the antibody of VAAs in the Western blot analysis and ELLA system. We also investigated the synergistic effects of the components in mistletoe by dividing the extract into different molecular weight fractions.

Antineoplastic Agents, Phytogenic↗

Study of the activity of differentiation-induction in a Chinese herb-viscum alniformosanae. Part 1: In vitro induction of differentiation in HL-60 leukemic cell line by conditioned medium secreted from viscum alniformosanae-stimulated mononuclear cells.

A conditioned medium(CM), designated as 572-CMF-, was a Chinese herb viscum alniformosanae (V.A.) stimulated mononuclear cells. This CM has the capacity to induce the promyelocytic cell line HL-60 to differentiate into morphologically and functionally mature monocytoid cells. However, our results on the effect of a combination of 572 conditioned medium and IFN-r, TNF and IL-2 were neither synergistic nor additive. Further investigation of the nature of this conditioned medium remains to be performed.

Cell Differentiation↗

Study of the activities of Chinese herb Viscum alniformosanae Part II: The components of conditioned medium produced by Viscum alniformosanae-stimulated mononuclear cells.

The human promyelocytic cell line HL-60 can be induced to monocytoid terminal differentiation by several conditioned media produced by lectin-stimulated mononuclear cells. We reported previously that a 572-conditioned medium (CM) secreted from viscum alniformosanae-stimulated mononuclear cells also had the capacity of inducing HL-60 leukemic cells into mature monocytes. In the present study, we showed that 572-CM did not contain IFN-r, TNF, IL-1 and IL-2 as determined by using ELISA tests. This CM was unable to induce granulocyte-macrophage colony formation. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the components of this CM. After running the acrylamide gels, a wide band protein, in the 65-80 kd range was obtained and it was different from those of other mitogens.

Cell Differentiation↗

Mechanisms involved in spontaneous and Viscum album agglutinin-I-induced human neutrophil apoptosis: Viscum album agglutinin-I accelerates the loss of antiapoptotic Mcl-1 expression and the degradation of cytoskeletal paxillin and vimentin proteins via caspases.

Viscum album agglutinin-I (VAA-I) is a plant lectin that possesses interesting potential therapeutic properties and immunomodulatory activities. We have recently found that VAA-I is a potent inducer of human neutrophil apoptosis, but the mechanism(s) involved require further elucidation. In this study, we found that VAA-I alters mitochondrial transmembrane potential and increases intracellular levels of reactive oxygen species (ROS). Despite these observations, treatment with the mitochondrial stabilizer, bongkrekic acid, or with catalase, known to degrade H(2)O(2), fails to reverse VAA-I-induced apoptosis. Moreover, VAA-I was found to induce apoptosis in PLB-985 cells deficient in gp91(phox), indicating that the lectin acts via an ROS-independent mechanism. Pretreatment of neutrophils with brefeldin A, an inhibitor of vesicular transport, was found to reverse VAA-I-induced apoptosis. Protein expression of Mcl-1 was decreased by VAA-I. The role of caspases in the degradation of cytoskeletal proteins during both spontaneous and VAA-I-induced neutrophil apoptosis was also investigated. Paxillin and vimentin were markedly degraded by VAA-I when compared with neutrophils that undergo spontaneous apoptosis, but not vinculin or alpha- and beta-tubulin. Caspases were involved in cytoskeletal protein degradation because preincubation with the pan-caspase inhibitor N-benzyloxycarbonyl-V-A-D-O-methylfluoromethyl ketone was found to reverse protein cleavage. We conclude that VAA-I needs to be internalized to mediate apoptosis and that its activity is not dependent on a cell surface receptor-mediated pathway. Also, we conclude that VAA-I induces apoptosis by ROS-independent and Mcl-1-dependent mechanisms and that caspases are involved in cytoskeletal protein degradation in both spontaneous and VAA-I-induced neutrophil apoptosis.

Apoptosis↗

Comparison of the effects of Viscum album lectin ML-1 and fresh plant extract (Isorel) on the cell growth in vitro and tumorigenicity of melanoma B16F10.

Numerous findings indicate that specific plant lectins acting against cancer could be major active components of Viscum album extracts, although activity of low molecular weight components (peptides, carbohydrates and alkaloids) might be as essential for the beneficial activity of the plain plant extracts, too. Thus, active principle of Viscum album extracts is still not understood, and is difficult to be analysed because of the complex composition of the extracts and uncertainty of the standardised effectiveness (batch consistency) of the extracts. The aims of this study were to compare the concentration dependent effects of the pure mistletoe lectin (ML-1) with the fresh plant Viscum album extract (Isorel) and its different MW components on the in vitro growth of ConA stimulated lymphocytes, on the growth and tumorigenicity (artificial lung metastases development) of murine melanoma B16F10 cells, and to compare concentration dependent effects of the different types of the Viscum album extracts in vitro (applying novel type of MTT assay). The results obtained indicate that the effects of Isorel used at high dose could be result of toxic activity of the mistletoe lectins ("ML-1 like" activity). Unlike ML-1, if used at low concentrations, Isorel selectively inhibited tumor cells, due the activity of the low MW components. On the other hand, the number of tumor nodules was reduced (in comparison to the control) equally in the lungs of mice injected with B16F10 cells pre-treated in vitro with the plain Viscum album extract or any of its modifications or ML-1. Hence, it is supposed that the beneficial therapeutic effects of Isorel might result from the combined biological activity of the high and the low MW components not lectins only. Similarly, in MTT assay low concentrations of all types of the Viscum album extract showed stronger inhibiting activity for B16F10 and HeLa cells than pure ML-1. According to these results we propose a standardisation of aqueous Viscum album extracts by comparing their and ML-1 concentration dependent activity on the tumor cells in vitro applying MTT bioassay described which should be relevant for further evaluation of their active principle and for improvement of biotherapy of cancer.

Adjuvants, Immunologic↗

Immunomodulatory effects of Viscum album agglutinin-I on natural immunity.

In 24 h cultured human peripheral blood mononuclear cells, treated with various (1 microgram/ml to 1 ng/ml) concentrations of Viscum album agglutinin-I, quantitative assessment of DNA breaks labelled with terminal deoxynucleotidyl transferase revealed a dose-dependent Viscum album agglutinin-I-induced apoptosis above a lectin concentration of 10 ng/ml. After 24 h incubation of peripheral blood mononuclear cells with non-cytotoxic concentrations of Viscum album agglutinin-I (10 and 1 ng/ml), messenger (m)RNA expression and secretion of a panel of cytokines were evaluated by reverse polymerase chain reaction and by enzyme-linked immunosorbent assay (ELISA), respectively. The lectin induced expression of interleukin-1 alpha, interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, interferon-gamma, granulocyte-monocyte colony stimulating factor and interleukin-10 genes, but no expression of interleukin-2 or interferon-gamma production could be detected. In addition, cellular components of the natural immune system (such as monocytes and granulocytes) bound Viscum album agglutinin-I molecules to a higher degree than lymphocytes. To establish the modulatory potency of Viscum album agglutinin-I on the natural immunity of human subjects, four randomized, double-blind crossover trials were performed on healthy volunteers. In contrast to the significant lectin-induced increases in number and activity of natural killer cells observed in animal models, in the first and second trial human healthy individuals showed no significant differences between their natural killer responses following an injection of lectin-enriched preparation or saline. Due to considerable intrinsic fluctuation of these parameters, a third and fourth double-blind trial with freshly isolated Viscum album agglutinin-I was performed using a more rapidly detectable parameter, the priming of granulocytes. Here, significant lectin-induced increases were found.

Agglutinins↗

Paediatric medulloblastoma cells are susceptible to Viscum album (Mistletoe) preparations.

BACKGROUND: Medulloblastoma constitute more than 20% of all paediatric brain tumours and are the most common malignant brain tumours in children. Adjuvant chemotherapy has seen a strong increase in the use of complementary medicine for cancer treatment. Evidence for cytotoxic and apoptotic effects of Viscum album (Mistletoe) in vitro is available, however, no data concerning paediatric tumours, especially paediatric brain tumours, has been provided so far. MATERIALS AND METHODS: In order to compare the receptiveness of medulloblastoma cells to different Viscum album preparations, in vitro cytotoxic effects of eight Viscum album extracts on four different paediatric medulloblastoma cell lines were analysed by MTT-Tests. Lectin contents of the extracts were determined to correlate them with the mitochondrial activity of mistletoe-treated cells. Flowcytometric analyses with Annexin V-FITC staining were carried out to quantify the amount of apoptotic cells compared to necrotic and viable cells. RESULTS: Data obtained with the medulloblastoma cell lines, Daoy, D342, D425 and UW-288-2, treated with Viscum album preparations from eight dissimilar host trees (Iscucin Abietis, Pini, Populi, Mali, Salicis, Crataegi, Quercus and Tiliae), indicated a significant growth-inhibition of all cell lines, yet the cell susceptibility was dissimilar against the different extracts. The decrease in mitochondrial activity and increase in apoptosis correlated with the lectin content of the used preparation in a dose-dependent manner. CONCLUSION: These in vitro results show that paediatric medulloblastoma cells respond to Viscum album preparations, by undergoing cell death through apoptosis and that this growth-inhibition correlates with the lectin content of the used preparation.

Annexin A5↗

Immunomodulatory and clinical effects of Viscum album (Iscador M and Iscador P) in children with recurrent respiratory infections as a result of the Chernobyl nuclear accident.

Ninety-two children 5 to 14 years of age living in areas exposed to the radioactive fallout from Chernobyl with recurrent respiratory infections (RRIs) were treated after randomization with either Viscum album praeparatum mali or pini (Iscador M or P). The dosage was two subcutaneous injections a week for 5 weeks with individual doses of 0.001 mg to 1.0 mg. Both Viscum album preparations were effective in significantly reducing clinical symptoms. One year after a single treatment course, the frequency of RRI relapses decreased by 78% and 73%, respectively. Immunomodulatory effects were assessed by investigation of lymphocyte subsets, natural killer (NK) cell activity, phagocytic and oxidative activity of polymorphonuclear leukocytes, and antiviral activity of serum before and 1 week after treatment. Viscum album therapy resulted in normalization of initial immune indices either below or above the normal ranges. High levels of antiviral activity before treatment were significantly decreased by Viscum album mali. Viscum album treatment should be studied further in children with RRI.

Adolescent↗

Seasonal and spatial variation of carbohydrates in mistletoes (Viscum album) and the xylem sap of its hosts (Populus x euamericana and Abies alba).

In the present field study we analysed the seasonal pattern of carbohydrate composition and contents in the xylem sap of Viscum album and the xylem sap of a deciduous (Populusxeuramericana) and a coniferous (Abies alba) host tree species. The results were compared with the soluble carbohydrate composition and contents of mistletoe tissues. On both hosts significant amounts of glucose, fructose, and sucrose were found in the xylem sap of Viscum throughout the seasons. The general seasonal pattern of sugar contents, i.e. high concentrations in spring and lower concentrations in other seasons on Populus, and intermediate concentrations throughout the year on Abies, largely reflected the xylem sap carbohydrate composition and contents of the respective host. These observations provide indirect evidence for carbohydrate flux from the xylem sap of the host into the mistletoe. However, in both hosts xylem sap seems to be deviated into the mistletoe without specific control of carbohydrate flux. Differences observed between the seasonal pattern of xylem sap carbohydrate concentrations in Viscum on Populus and Abies may originate from the different time of leaf development of these species. A clear-cut seasonal pattern of soluble carbohydrates was not observed in the leaves of Viscum on both hosts. Still soluble carbohydrates seem to be reallocated from the senescing to the newly developed leaves of Viscum indicating that the seasonal requirement of carbohydrate for growth and development can only completely be met by carbohydrate acquisition from the host and their own photosynthesis.

Journal Article↗

Phase II study of viscum fraxini-2 in patients with advanced hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Although a wide range of therapeutic options is available, the efficacy of these methods and the prognosis of patients with HCC remain very poor. This study was conducted to evaluate the efficacy and safety of viscum fraxini-2 in patients with chemotherapy-naïve, advanced hepatocellular carcinoma. 23 patients with unrespectable HCC who had received no prior systemic chemotherapy with objectively measurable tumors were enrolled on this study. The mistletoe preparation for the study is an aqueous injectable solution. It contains one milliliter of viscum fraxini in dilution stage-2 (15 mg extract of 20 mg mistletoe herb from ash tree, diluted in di-natrium-mono-hydrogen phosphate, ascorbic acid and water) which is equivalent to 10 000 ng/ml injection ampoules. 2 ampoules of viscum fraxini-2 were administered subcutaneously once weekly. As assessed by conventional imaging criteria, 3 (13.1%) patients have achieved complete response, 2 (8.1%) patients have achieved a partial response. 9 (39.1%) had progressive disease while 9 (39.1%) patients didn't have evaluation of response due to early death. The median overall survival time for all patients was 5 months (range 2-38 months), for those who achieved a CR was 29 months (range 12-38 months) and, for those who achieved a PR was 6.5 months (range 6-7 months). The median progression free survival for all patients was 2 months (range 1-38 months), for those who achieved a CR, it was 29 months (range 8-38 months) and for those who achieved a partial response, it was 5 months (range 4-6 months). No hematologic toxicity has been encountered. The spectrum of non-hematologic toxicity was mild. The WHO toxicity criteria grade 3-4 were 34.8% drug related fever, 13.1% erthyma at injection site and 17.4% pain at the site of injection. No drug related discontinuation or toxic deaths have occurred. Viscum fraxini-2 seems to be particularly promising in patients with advanced HCC, it shows antitumor activity and low toxicity profile. Further studies in combination with other active agents are clearly warranted.

Adult↗

Influence of the parasite Viscum cruciatum Sieber on the chemical constituents of Crataegus monogyna Jacq.

A phytochemical study of two plant species, Viscum cruciatum Sieber and Crataegus monogyna Jacq., was completed to investigate the influence of the parasite Viscum cruciatum on the host Crataegus monogyna. The study was carried out with two samples and consisted of hexane extracts of the Viscum cruciatum parasitizing on Crataegus monogyna and C monogyna. In these samples ursolic acid, beta-sitosterol and a triterpene fraction were found that contained mainly butyrospermol (3beta-lanost 8, 24-dien, 3-ol), 24-methylene-24-dihydrolanosterol (24-methylene-5alpha-lanost-8-en-3beta-ol), cycloartenol (9beta, 19-cyclo-5alpha, 9beta-lanost-24-en-3beta-ol), beta-amyrin (olean-12-en-3beta-ol) and several aliphatic alcohols identified as the C18 to C30 members of the 1-alkanol homologous series. beta-Amyrin acetate was only isolated from Viscum cruciatum and was not found in Crataegus monogyna.

Alcohols↗

Structure and properties of polysaccharides from Viscum album (L.).

Polysaccharides are possibly involved in the pharmacological effects of Viscum album (mistletoe) extracts, which are used in cancer therapy. Therefore the water-soluble polysaccharides of the fresh plant and the fermented proprietary preparation Iscador were isolated and characterized inter alia by methylation analysis, partial hydrolysis and C-13-NMR spectroscopy. The main polysaccharide of the green parts of Viscum is a highly esterified galacturonan whereas in Viscum 'berries' a complex arabinogalactan is predominant. Both types of these constituents were found in Iscador but with definite changes in molecular weight and structure. An interaction between the arabinogalactan and the galactose-specific lectin (ML I) in Viscum could be demonstrated. In three immunological tests (granulocyte, chemiluminescence, carbon clearance test) the polysaccharides failed to increase phagocytic activity of granulocytes and macrophages.

Carbon↗

Reduction of leukopenia in mice by "viscum album" administration during radiation and chemotherapy.

"Viscum album" extract, iscador, was found to reduce the leukocytopenia produced by radiation and cyclophosphamide treatment in animals. Weight loss due to radiation was considerably reduced by "Viscum album" extract whereas weight loss due to cyclophosphamide was not altered. Hemoglobin levels also were not affected by "Viscum album" extract administration. The results indicated that treatment with "Viscum album" extract reduces lymphocytopenia and hence could be used along with chemotherapy and radiation therapy.

Animals↗

Are lectins of Viscum album interesting tools in lung diseases? A review of recent results.

The data presented provide evidence that lectins isolated from different sources such as Abrus and Ricinus seeds and Viscum leaves are structurally related proteins and are probably homologues. The lectins have overall similarities in physical, chemical and biological properties. These proteins appear to be immunologically closely related and possess a high degree of conservation in their primary structures, anticipating a role in fundamental pathways common to all plants. Methods for the preparation, purification and characterization of the Viscum album lectins and comparative crossreactions with closely related lectins of the "toxic lectin" group are summarized. The biological properties of Viscum album lectins, especially their reaction with phagocytes, (phagocytosis), basophil granulocytes (histamine release) and tumour cells (inhibition of tumour growth) are presented and compared with those obtained with an anti-tumoral preparation derived from Viscum album, ISCADOR.

Amino Acids↗

Molecular characterization of the recombinant A-chain of a type II ribosome-inactivating protein (RIP) from Viscum album coloratum and structural basis on its ribosome-inactivating activity and the sugar-binding properties of the B-chain.

Mistletoe (Viscum album) lectins, which are classified as a type II ribosome-inactivating protein (RIP) due to their unique biological function and the potential medical and therapeutic application in cancer cells, receive a rising attention. The heterodimeric glycoproteins contain the Achain with catalytic activity and the B-chain with sugar binding properties. In recent years, studies involving the lectins from the white berry European mistletoe (Viscum album) and the yellow berry Korean mistletoe (Viscum album coloratum) have been described. However, the detailed mechanism in exerting unique cytotoxic effect on cancer cells still remains unclear. Here, we aim to understand and define the molecular basis and biological effects of the type II RIPs, through the studies of the recombinant Korean mistletoe lectin. To this end, we expressed, purified the recombinant Korean mistletoe lectin (rKML), and investigated its molecular characteristics in vitro, its cytotoxicity and ability to induce apoptotic cell death in cancer cells. To gain structural basis for its catalytic activity and sugar binding properties, we performed homology modeling studies based on the high degree of sequence identity and conserved secondary structure prediction between Korean and European, Himalayan mistletoe lectins, and Ricin.

Amino Acid Sequence↗

[Isolation and determination of homoeriodictyol-7-O-beta-D-glycoside in Viscum coloratum].

The homoeriodictyol-7-O-beta-D-glycoside was isolated from Viscum coloratum and identified by mass spectrometry and nuclear magnetic resonance (NMR) (1H NMR and 13C NMR). A method for determination of homoeriodictyol-7-O-beta-D-glycoside in Viscum coloratum was developed by using a Kromasil C18 column (200 mm x 4.6 mm i.d., 5 microm) with a mixture of acetonitrile and 0.5% glacial acetic acid solution (18:82, v/v) as mobile phase at a flow rate of 1.0 mL/min. The detection wavelength was set at 284 nm and temperature was set at 30 degrees C. The volume of injection was 10 miccro L. Good linear relationship (r = 0.9997) between the mass concentration and the peak area of homoeriodictyol-7-O-beta-D-glycoside was obtained in the range of 1.0-32.0 mg/L. The recoveries were found to be in the range of 96.0%-100.1%. The results of the experiments demonstrated that the established method is rapid and simple with good accuracy and reproducibility. The method is suitable for the quality control of Viscum coloratum from different sources.

Flavonoids↗