Viscum Album Research Articles

Antitumoral activity of homeopathic and anthroposophic Viscum album's preparations: an in vitro assay

Background: Viscum album (mistletoe) has been used for a variety of purposes and is widely used as an alternative treatment for cancer and other diseases. Preparations of fermented extracts from Viscum album (VA), a plant from the Loranthaceae family, have been used, mainly in European countries, with promising results as adjuvant therapies, especially in Anthroposophic Medicine.1,2,3. In vitro studies have demonstrated that various types of VA may have cytotoxic activity in carcinoma cells, being able to activate the apoptotic cascade or leading cells to necrosis. It is possible to prepare VA extracts using different procedures and solvents. The homeopathic preparations involve the use of ethanol as solvent4, while the antroposophic preparations are obtained in aqueous solvent5. 
 Aims: The present study aimed to verify the cytotoxic effects of homeopathic and anthroposophic Viscum album preparations in human leukaemic K562 cell line. 
 Methodology: The cytotoxic effects of anthroposophic (ISCADOR A - IA; ISCADOR P - IP; ISCADOR M - IM; ISCADOR U - IU; ISCADOR Qu - IQu) and homeopathic Viscum album extracts (VA) were evaluated in K562 cells. For this, ten independent experiments of Trypan blue assay and five independent MTT assay were carried out.6 Additionally, cell morphology was analysed by phase inverted contrast microscopy coupled to digital camera (Optika XDS-3).7 The results were expressed as mean±SD, and statistical comparisons were performed by one-way ANOVA. 
 Results: All samples tested were able to interfere with viability and mitochondrial activity, with important morphological damage, when compared to respective controls. Considering the ISCADOR samples all of them presented similar cytotoxic activity, with lower activity attribute to IP (around 25% of viable cells) in comparison to 90% of non-viable cells induced by other samples. The homeopathic preparation originated the best results compared to anthroposophic samples. Morphological analyses supported the MTT and trypan blue assays´ results. 
 Conclusion: The anticancer activity tested in K562 indicate that all Viscum album samples have anticancer activity, in a dose dependent manner. Further tests need to be done in order to contribute for the understanding of the Viscum album antitumoral mechanisms, as well as about differences between homeopathic and antroposophic procedures.

Two cases reports comparing histopathological features of canine transmissible venereal tumor after treatment with decimal potencies of Viscum Album and Vincristin

The aim of the study was to evaluate the histopathological features of two canine transmissible venereal tumor (TVT) cases: one treated with Viscum album (Q) protocol in serial decimal potencies and the other with Vincristin conventional protocol (treated control). Tumor infiltrating leukocytes pattern and apoptosis were analyzed quantitatively, using a computer image analysis system, twice: before the beginning of treatment and after six weeks. Both female dogs were sujected to weekly clinical evaluation for three months, focusing on tumor remission and eventual adverse effects manifestations (local or systemic). In both the cases, animals had a round cells tumor growth, with weak intercellular stroma connection, typical of a TVT mass. The lysozyme positivity in immunohistochemistry analysis, associated with typical morphology confirmed the diagnosis. Ten fields were randomly photographed by a digital photomicrography system NIKON Eclipse 2 – Coolpix. Each captured image was analyzed quantitatively by the Metamorph® software, considering the number of leukocyte infiltrated and mitosis per field. Caspase 3 positivity index was also evaluated. In Vincristin treated dog, the number of leukocytes infiltrating tumor was bigger than that found in Viscum album treated dog, even before the beginning of the treatment, indicating an initial conditions compatible to a more favorable prognosis, however, the number of leukocytes increased in the Viscum album treated dog during the observation period (Kruskal Wallis, p < 0.05). The animal treated with Vincristine has no statistical significant variation in the number of infiltrating mononuclear cells during the treatment, but presented 10 times more mitotic figures (Kruskal Wallis, p < 0.001) in the second biopsy, compared to the other stages of treatment. There was a statistical significant increase in the rate of positivity for Caspase 3 between biopsies taken before and after treatment with Viscum album (ANOVA, p=0.01), but not after Vincristin treatment. Even though, the tumor remission could be achieved only in Vincristin treated dog, with an abrupt reduction of mitosis number and tumor mass in the last 20 days of treatment. In contrast, a secondary infection of the tumor led to clinical worsening of TVT in Viscum album treated dog, which could explain the increase in the local leukocytes number. The increase in Caspase 3 positivity after treatment shows the Viscum album anti-neoplastic mechanism of action, but in this case it was not enough to reach a complete tumor remission.

Viscum album (L) extracts in cancer treatment: a systematic review of in vitro and in vivo studies

Background: Several reviews about phytotherapy and homeopathy have been published in the last years, including Viscum album (VA.L). VA is a parasite plant whose extract has anti-cancer proprieties and is used alone or in combination with conventional chemotherapy. 
 Methods: We performed a systematic review about the in vivo and in vitro models described in the literature, including veterinary clinical trials. The literature was consulted from Pubmed database.
 Results: There are several kinds of pharmaceutical preparations about VA and their active principles used in experimental studies, lectin being frequently studied (alone or as an extract compound). More than 50% of available literature about VA is related to the lectin effects. On the other hand, the effects of viscotoxins are less studied. Among the in vivo experimental studies about VA and its compounds, the B16 murine melanoma is the most used model, followed by Ehrlich, Walker and Dalton tumors. The results point to the apoptotic effects, metastasis control and tumor regression. Some veterinary clinical studies about the use of VA in the treatment of sarcoid, fibrosarcoma and neuroblastoma are quoted in literature too, with interesting results. Considering the in vitro models, our review revealed that NALM6 leukemia cells, B16 melanoma and NC1-H460 lung carcinoma were the most studied tumor models, apoptosis signals being the most important findings. Only one study verified immunoglobulin and interleukin production. All consulted papers were related to phytotherapy preparations only.
 Conclusions: Although the literature about the anti-cancer activity of VA extract and its lectins is enough, there is a marked lack of information about viscotoxin activities and about the effects of homeopathic preparations of this plant on animal tumors and on in vitro cultivated tumor cells.

Metabolomics by UHPLC-Q-TOF Reveals Host Tree-Dependent Phytochemical Variation in Viscum album L.

Viscum album L., commonly known as European mistletoe, is a hemi-parasitic plant of the Santalaceae family. The in vitro and in vivo effects of V. album differ, according to its host tree. However, little is known about the host-dependent phytochemical diversity in V. album. In this study, the metabolic profiles of V. album ssp. album from Malus domestica Bork., Quercus robur L., and Ulmus carpinifolia Gled were compared. Leaves, stems, and berries were collected in Switzerland, by the same procedure, in September 2016 and 2017. The methanolic extracts were analyzed by ultra-performance liquid chromatography, coupled to electrospray quadrupole time-of-flight mass spectrometry in positive ionization mode. The data were submitted to partial-least square discriminant analysis (PLS-DA) and the results showed that the V. album ssp. album samples were clustered into three groups, according to the three distinct host trees. Seven compounds, with high VIP scores (variable importance in projection), were responsible for this differentiation. The following four compounds were detected in both the harvest years: arginine, pipecolic acid or lysine, dimethoxycoumarin, and sinapyl alcohol, suggesting their use as host specific V. album biomarkers. The present work highlights the importance of standardized harvest and analytical procedures for the reproducibility of the chemical results of herbal materials.

Viscum album : from high dilutions to phytotherapy

Background : Besides conventional anticancer therapy, cancer patients use complementary and alternative medicine like the medicinal herb mistletoe (Viscum album L.), used in oral form or injections.
 For this condition Viscum album (VA) is also used in high dilutions, in injections, as in Brazil.
 
 Aim:
 Our aim is to compare results of basic research about these two presentations: herbal medicine and high dilutions and to look for an eventual continuity of action between the molecular form (herbal medicine) and the informative one (high dilutions)
 
 Method:
 About VA in herbal medicine : a lot of references in immunology, in particular those published by the team of Srini Kaveri and Stephan Baumgartner.(1) (2) (3) (4) (5)
 About VA in high dilutions : the work in immunology of Aloiso Cunha de Carvalho and Leoni Bonamin published in a thesis. (6)
 And then to compare those results with our practice as veterinarian on dogs and cats .
 
 Results : 
 VA in herbal medecine: from basic research results, we know that in low concentration, 
 an immunostimulary action is observed while in higher doses it is a cytotoxic activity.
 These two properties are found in our practical use of VA in dogs or cats : stabilization of the size of tumors for the low concentrations, necrosis of tumors and abscess for the high concentrations. 
 VA in high dilutions (from D3 to D30) : from basic research, an immunostimutating effect is observed. More exactly, on a murin model of tumor of Ehrlich, it is observed an improvement of quality of life, a reduction of the tumor immunosuppressive effect, an enlargement of local inflammations (Oedema) for all concentrations, but only 3D reduces tumor growth, without modifying local necrosis and the immunological parameters.
 In our practice, on two cases of osteosarcoma on dogs, we verify those properties using VA both in herbal medicine and in high dilutions. 
 
 Conclusions:
 From these results, both in basic research and in the practice, we should estimate there is probably a continuity of action fom VA, in High Dynamised Dilutions ( no more molecule), to VA in herbal medicine (molecular activity). From immunostimation to cytotoxicity, from information to molecular activity.
 
 Does each Viscum album’s preparation touch
 the same level of the Immune System ? The same cells ? The same receptors ? A lot of questions remain…
 
 
 References 
 
 
 1) Duong Van Huyen JP and al. Variable sensitivity of lymphoblastoid cells to apoptosis induced by Viscum album Qu FrF, a therapeutic preparation of mistletoe lectin. Chemotherapy. (2001)
 
 2) Duong Van Huyen JP and al. Induction of apoptosis of endothelial cells by Viscum album: a role for anti-tumoral properties of mistletoe lectins. Mol Med. (2002)
 
 3)Duong Van Huyen JP and al Comparative study of the sensitivity of lymphoblastoid and transformed monocytic cell lines to the cytotoxic effects of Viscum album extracts of different origin.. Chemotherapy. (2003)
 
 4) Estko M and al. Tumour cell derived effects on monocyte/macrophage polarization and function and modulatory potential of Viscum album lipophilic extract in vitro.
 BMC Complement Altern Med. 2015
 
 
 5) Saha C, and al Differential Effects of Viscum album Preparations on the Maturation and Activation of Human Dendritic Cells and CD4⁺ T Cell Responses.
 Molecules. 2016 Jul 14;21(7)
 
 6) Aloisio Cunha de Carvalho , ATIVIDADE ANTI-NEOPLÁSICA DE Viscum album (L) EM TUMORES EXPERIMENTAIS: REVISÃO CRÍTICA E ESTUDO EXPERIMENTAL EM TUMOR DE EHRLICH, Thesis Sao Paulo

Green Synthesis of Silver Nanoparticles Using Natural Extracts with Proven Antioxidant Activity.

Natural extracts are a rich source of biomolecules that are useful not only as antioxidant drugs or diet supplements but also as complex reagents for the biogenic synthesis of metallic nanoparticles. The natural product components can act as strong reducing and capping substrates guaranteeing the stability of formed NPs. The current work demonstrates the suitability of extracts of Camellia sinensis, Ilex paraguariensis, Salvia officinalis, Tilia cordata, Levisticum officinale, Aegopodium podagraria, Urtica dioica, Capsicum baccatum, Viscum album, and marine algae Porphyra Yezoensis for green synthesis of AgNPs. The antioxidant power of methanolic extracts was estimated at the beginning according to their free radical scavenging activity by the DPPH method and reducing power activity by CUPRAC and SNPAC (silver nanoparticle antioxidant capacity) assays. The results obtained by the CUPRAC and SNAPC methods exhibited excellent agreement (R2~0.9). The synthesized AgNPs were characterized by UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), dynamic light scattering (DLS) particle size, and zeta potential. The UV-vis absorption spectra showed a peak at 423 nm confirming the presence of AgNPs. The shapes of extract-mediated AgNPs were mainly spherical, spheroid, rod-shaped, agglomerated crystalline structures. The NPs exhibited a high negative zeta potential value in the range from −49.8 mV to −56.1 mV, proving the existence of electrostatic stabilization. FTIR measurements indicated peaks corresponding to different functional groups such as carboxylic acids, alcohol, phenol, esters, ethers, aldehydes, alkanes, and proteins, which were involved in the synthesis and stabilization of AgNPs. Among the examined extracts, green tea showed the highest activity in all antioxidant tests and enabled the synthesis of the smallest nanoparticles, namely 62.51, 61.19, and 53.55 nm, depending on storage times of 30 min, 24 h, and 72 h, respectively. In turn, the Capsicum baccatum extract was distinguished by the lowest zeta potential, decreasing with storage time from −66.0 up to −88.6 mM.

Safety Assessment of Intravenous Application of the Homeopathic Medicine Viscum Album D3 in Dogs

Background: Viscum album (VA) extracts have a wide variety of biologically active compounds that can be used in the treatment of various diseases, with cancer being the main one. However, few data on the effects of homeopathic VA are found in the literature, even though commercially VA formulations are available, with extremely low doses of its active compounds. Previous studies used the anthroposophical form of Viscum and provided information on the mechanisms of action of this drug in tumor cells or in cells of the immune system. Materials and methods: This study aimed to evaluate the safety of intravenous and subcutaneous application of D3 homeopathic Viscum album (VAD3) in dogs. Healthy dogs (n=7) underwent blood collection for blood cells count and biochemistry analysis and clinical examinations 7 days prior and at days 0, 3, 5, 7, 10, 12, and 14 after the beginning of treatment. The dogs received intravenous application of VAD3 at days 0, 5, 10, and 14, and subcutaneous at days 3, 7, and 12. Results: There was a transient increase in the number of monocytes (P=0.0022) at day 5, while there were no significant (P>0.05) changes in any of the other blood endpoints over time. Nevertheless, all blood parameters remained within the reference values for the species. All animals completed the study in good health conditions. According to the Quality-of-Life Assessment Questionnaire, all animals showed improvement in mood and appetite after receiving VAD3. Conclusion: The homeopathic medicine VAD3 is safe for intravenous and subcutaneous applications in dogs, besides possibly bringing benefits to the entire organism.

Inventorization of traditional ethnobotanical uses of wild plants of Dawarian and Ratti Gali areas of District Neelum, Azad Jammu and Kashmir Pakistan.

BackgroundAn ethnobotanical expedition was conducted to document the traditional ethnobotanical (TEB) uses of wild flora of Dawarian and Ratti Gali villages of District Neelam, Azad Jammu and Kashmir (AJK) Pakistan. District Neelam has rich plant diversity and is hub of many endemic plant species while the study areas are not yet explored. The research area: Dawarian and Rati Gali (DRG) area is mountaineous terrain and villages are located on far and farther distances. DRG area has rich biocultural and plant diversity comprising of different ethnic groups of Kashmir state. The current research was aimed to explore and document traditional medicines (TEMs) and other domestic and commercial uses of wild plants. This study will assist to evaluate conservation and commercial worth of wild flora which can be potential candidate for drug discovery through ethnopharmacological analysis.MethodsThe current quantitative ethnobotanical research was carried out in 2018 by interviewing 150 indigenous informants (90 male and 60 female) of DRG area using questionnaire applying structured and semi structured interview methodology. Data analysis was analyzed by using quantitative ethnobotanical statistical tools such as fidelity level (FL), informant consensus factor (ICF), Spearman’s rank correlation (SRC) and data matrix ranking (DMR).ResultsThe indigenous people of DRG area use wild plants in their daily life to cope life necessities i.e. food, vegetables, fodder, fuel, shelter, timber and herbal medicines. TEMs are primarily used to cure different infirmities like diabetics, asthma, dysentery, constipation, cold, fever, joint pain, wound healing, cancer, cardiovascular disorders, epilepsy, kidney infections and many types of skin diseases. Current study revealed the data of 103 wild plants species belonging to 46 plant families from selected areas of District Neelum, AJK. Results depicted that Asteraceae ranked 1st (12 plants spp). Among plant parts used leaf ranked 1st (18%), followed by seed (17%) and root (13%). While prevalent form recipe mode was decoction (20%), followed by powder (17%) and extract (14%) and fodder was highest (37%) EB use-form fodder, followed by food (32%) and fuel (17%). Quantitative ethnobotanical analysis (QEA) was carried to find the reliability and novelty of the study. Five plant species including Berberis lyceum (FL = 97.78%), Isodon rugosus (FL = 95.71%), Saussurea lappa (FL = 94.74%), Aconitum heterophyllum (FL = 92.71%) and Taxus baccata (91.58%) had shown high fidelity level which confirmed that these plants have high medicinal worth in study area. The highest value (0.94) of ICF was for diseases group “tuberculosis and leucorrhea”, followed by stomachache and flatulence (0.93), diabetics and blood pressure (0.92) and asthma and chest infections (0.88). For other uses fuel with ICF (0.83) ranked first and second was hedging and thatching (ICF = 0.82) where people use plants or their parts for construction. Spearman’s rank correlation (SRC) test indicated that number of TEB uses increases if number of species is increased. Jaccard index (JI) analysis depicted that 56.31% plants are being used as TEMs which are first time explored from the study area. While 26.21% plants are being used in different TEB uses which are different from past cited literature. These novel findings of research indicate that wild flora of the study area has great potential for novel drug discovery and provision of materialist services for the indigenous communities.ConclusionThe present research revealed that TEMs uses of 58 plants are novel being first time reported from the study area (DRG) of District Neelam of AJK. The results showed that plants like Acer cappadocicum, Ajuga bracteosa and Swertia paniculata are used to cure diabetes, Viscum album, Viola canescens, Taxus baccata are used for cure of cancer, Isodon rugosus, Polygala chinensis are used in TEMs for treating cardiovascular disorders and Anaphalis triplinervis is used for epilepsy. Berberis lyceum, Ajuga bracteosa, Aconitum heterophyllum, Bistorta amplexicaule, Saussurea lapa and Jurinea dolomiaea are severely threatened and there is urgent need to do conservation measures for available of valuable MPs to the indigenous communities for life necessities and for future research. The current study will also be useful addition in ethnobotanical database, preservation of traditional culture and drug discovery and drug development through future ethnopharmacological research.

Biological activity and phytochemical analysis of homeopathic preparations of Viscum album L

Several in vitro and in vivo studies investigated the biological properties of mistletoe preparations [1-4]. In Europe, where phytochemical constituents of aqueous Viscum album extracts were previously described, the most widespread and common form of mistletoe is the white-berried species (Viscum album) [5]. The aim of the present study was to analyze the biological activity and phytochemical features of different Viscum album ssp mother tinctures collected in 2 different harvesting periods. 
 
 Leaves, stems and berries of Viscum album samples were subjected to ethanolic extraction (45% v/v) following the homeopathic pharmacopeia [6,7]. The mother tinctures (MT) prepared were: V. album (from 3 host trees Malus domestica, Quercus sp, Ulmus sp), V. album ssp. austriacum (host tree Pinus sylvestris) and V. album ssp. abietis (host tree Abies pectinata). All samples were collected at Höfli farm (Switzerland) in July and August and subjected to chemical analysis by means of high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) and Thin-Layer Chromatography (TLC). Tumor (Yoshida and Molt4) and non-tumor cells (Ma104) were incubated with MT (1; 0.1; 0.01%) for 4, 24 and 48 hours at 37ºC. Proliferation was indirectly measured using WST-1 (tumor cells) and MTT (non-tumor cells). All cells (1.5 x 104/100 μL) were plated in triplicate in 96-well plates. The cell proliferation inhibition rate was calculated as percent inhibition relative to control cells treated with the MT solvent (45% ethanol v/v). Apoptosis/necrosis was measured using 2 x 105 cells incubated with Annexin V-FITC and 7-AAD at room temperature in the dark, and analyzed in flow cytometer [2]. Antimicrobial activity was assessed for Candida albicans, Cryptococcus neoformans, Escherichia coli and Staphylococcus aureus following the Clinical Laboratory Standards Institute manuals [8-9] and compared with 45% ethanol v/v (MT solvent).
 
 The chromatographic assays of MT samples showed phenolic acids and lignans as main chemical classes identified. WST-1 and MTT experiments revealed different antitumor potential of MT. In general, Yoshida cells were more sensitive than Molt4. The maximal cytotoxic effect with 100% of mortality was attained by incubation of Yoshida and Molt-4 cells with 1% MT of V. album ssp. abietis (harvested in July) for 4 hours. Preliminary results with non-tumor cells indicate lower cytotoxicity compared to tumor cells. Annexin V/7ADD showed predominance of late apoptotic/necrotic events after 4 and 24 hours of incubation with all MT. The antimicrobial assay showed that MT of Viscum album ssp. austriacum (host tree Pinus sylvestris) exhibited minimum inhibitory concentration 7.25 mg/mL for the 2 tested bacterial strains. 
 
 The results of the present study suggest that Viscum album samples prepared according to the homeopathic pharmacopeia exhibit promising biological activities in a dose-dependent manner. The effects of potentiation will be assessed and compared to non-potentiated samples in future experiments. Therefore, the present study contributes with data on the main chemical components in homeopathic preparations of Viscum album, drawing attention to the relevance of extract solvent, seasonal aspects, host tree and harvested parts of plant harvested to the quality of thee mother tinctures.

Investigation of force-like effects of potentized preparations

Abstract
 The scope of this project is the investigation of the possible occurrence of force-like effects of potentized preparations. Based on earlier work [1, 2] we have chosen two preclinical models: a droplet evaporation method applied on wheat seeds and a bioassay with cress stressed with colchicine. In both methods we investigate if potentized and not potentized substances exhibit force-like effects. 
 We study two types of potentization procedures. First we study classical homeopathic potentization (Natrium muriaticum 30cH) and compare it with unpotentized NaCl 0.9%. Second, we study anthroposophic potentization (Iscador, mistletoe (Viscum album L.) extracts treated with a special mechanized pharmaceutical process) and compare it with unpotentized mistletoe extract (not mechanically treated).
 Droplet evaporation method is performed as described in detail elsewhere [1]. In short: wheat seeds were placed in the inner space of bilayer recipients, whereas, in the outer space (i) the unpotentized substance, (ii) the potentized substance, or (iii) a water control is placed. After one week wheat seeds are removed from the recipients and placed in water for 1h. Droplets of the obtained leakage were evaporated on glass-slides in controlled conditions. The droplet residues are photographed in magnification 100x and analyzed for fractal dimension.
 Cress growth test is performed as described in detail elsewhere [2]. For the study of force-like effects, ampoules of the above mentioned substances and ampoules of water for injection are placed for 2 days in recipients filled with purified water. Subsequently 20 µg/ml colchicine were added. The so prepared water is used to grow selected cress seeds in bags for 4 days. The plants grow in darkness under controlled conditions and show a morphological malformation due to the colchicine. The length of the root and shoot at each plant is measured and the root/shoot ratio is calculated, to determine if there is any difference between the treatments.
 
 1. Kokornaczyk, M.O., S. Baumgartner, and L. Betti, Preliminary study on force-like effects between As45x, water, and wheat seeds performed by means of the droplet evaporation method. International Journal of High Dilution Research, 2015. 14(2): p. 17-19.
 
 2. Baumgartner S, Flückiger H, Kunz M, Scherr C, Urech K. Evaluation of Preclinical Assays to Investigate an Anthroposophic Pharmaceutical Process Applied to Mistletoe (Viscum album L.) Extracts. Evid Based Complement Alternat Med. 2014: Article ID 620974.

Viscum album homeopathic tinctures: phytochemical profile and antiproliferative activity

Background: The mistletoe Viscum album L. (Santalaceae) is a semi-parasitic plant that grows on different host trees. Although V. album antitumor activity is mainly associated with aqueous preparations, recently we showed antitumoral effects of homeopathic V. album tinctures containing also the ethanolic soluble compounds [1]. 
 Aims: to analyze the phytochemical profile of different Viscum album homeopathic tinctures as well as their in vitro effects in tumoral cells. 
 Methodology: The Viscum album samples (leaves, stems and berries) were collected in 2016 and 2017 (summer), and in 2018 (winter) at different locations in Switzerland. After harvest, they were immediately submitted to ethanolic extraction (45% V/V) using homeopathic methodology [2, 3]. The following mother tinctures (MT) were prepared: V. album ssp. album growing on Malus domestica, Quercus sp. and Ulmus sp., V. album ssp. austriacum from Pinus sylvestris, and V. album ssp. abietis from Abies alba. The homeopathic dynamizations (1-3dH; 6, 12, 30dH) were prepared with the respective MT. The phytochemical profiles were analysed by High Performance Liquid Chromatography tandem Mass Spectroscopy, HPLC and Thin Layer Chromatography. MT prepared in 2016 were also submitted to the Pfeiffer’s circular chromatography (PCC) [4]. The proliferation assay was performed by WST-1 [5] after incubation of tumoral cells (Yoshida and Molt-4) with non-dynamized (0.5 to 0.05% v/v) and dynamized MT. Apoptosis/necrosis was measured by flow cytometer (FACS) using Annexin V-FITC [5], and the cellular morphological aspects were analysed by light microscope. 
 Results and Discussion: The chemical analyses of MT identified the presence of phenolic acids, flavonoids, lignans, as principal compounds. Besides, the HPLC indicated higher viscotoxin concentration in the summer harvest of Abies alba, Malus domestica and Quercus (897, 475, 219 ?g/mL), respectively. The PCC methodology permitted the MT differentiation. The higher levels of cellular mortality were attribute to Abies alba, Malus domestica and Quercus (p

Untargeted metabolomics approach to discriminate mistletoe commercial products

Mistletoe (Viscum album L.) is used in German-speaking European countries in the field of integrative oncology linking conventional and complementary medicine therapies to improve quality of life. Various companies sell extracts, fermented or not, for injection by subcutaneous or intra-tumoral route with a regulatory status of anthroposophic medicinal products (European Medicinal Agency (EMA) assessment status). These companies as well as anthroposophical physicians argue that complex matrices composed of many molecules in mixture are necessary for activity and that the host tree of the mistletoe parasitic plant is the main determining factor for this matrix composition. The critical point is that parenteral devices of European mistletoe extracts do not have a standard chemical composition regulated by EMA quality guidelines, because they are not drugs, regulatory speaking. However, the mechanism of mistletoe’s anticancer activity and its effectiveness in treating and supporting cancer patients are not fully understood. Because of this lack of transparency and knowledge regarding the matrix chemical composition, we undertook an untargeted metabolomics study of several mistletoe extracts to explore and compare their fingerprints by LC-(HR)MS(/MS) and 1H-NMR. Unexpectedly, we showed that the composition was primarily driven by the manufacturer/preparation method rather than the different host trees. This differential composition may cause differences in immunostimulating and anti-cancer activities of the different commercially available mistletoe extracts as illustrated by structure–activity relationships based on LC–MS/MS and 1H-NMR identifications completed by docking experiments. In conclusion, in order to move towards an evidence-based medicine use of mistletoe, it is a priority to bring rigor and quality, chemically speaking.

Abstract LB102: Effect of mistletoe extract on growth and proliferation of prostate cancer cells

Abstract Complementary and alternative medicines are explored for their role in improving the quality of life of patients with cancer. Many anti-cancer drugs have been derived from natural sources. Mistletoe extract, composed of lectins, viscotoxins, flavonoids, and other biologically active substances, has two mechanisms of action: tumor cytotoxicity and immunomodulation. The cytotoxic effects of mistletoe extract are a result of protein synthesis interference, cell cycle inhibition, and apoptosis induction. Further, mistletoe extract has been shown to have anti-angiogenic properties. In Europe, Helixor M, mistletoe extract from Viscum album mali, is used as an anthroposophic medicine in the treatment of malignant and non-malignant tumors, malignant disorders with accompanying impairment of the hematopoietic organs, stimulation of bone marrow activity, reducing the risk of tumor recurrence, and diagnosed pre-cancerous disorders. However, only limited evaluation of mistletoe extract has been conducted in the United States. We studied the effects of Helixor M in prostate cancer. To evaluate its effect on cell proliferation and survival, we treated androgen receptor (AR) positive (LNCaP, LAPC4 and VCaP) and AR negative (PC3 and DU-145) cell lines with Helixor M. Our data indicates that LNCaP and LAPC4 cells were most sensitive to Helixor M while PC3, DU-145, and VCaP cells were relatively resistant to treatment. While AR protein levels and NF-kappaB subunits remained unaffected by treatment, there was an induction of apoptosis along with a concomitant decrease in LC3B, an autophagy marker, in all the sensitive prostate cancer cell lines. In search of pathways that may synergize with growth inhibition by Helixor M, we utilized CRISPR knockouts of TBK1, a kinase involved in autophagy induction, in LNCaP cells and tested whether TBK1 knockouts are synthetically lethal with Helixor M. Helixor M induced increased apoptosis in TBK1 knockouts as compared to parental LNCaP cells, indicating that TBK1 loss might be synthetically lethal with Helixor M. Citation Format: Kavya Boyapati, Rajendra Kumar, Lillian Wilson, Yuhan Yang, Deven Topiwala, Keerti Soundappan, Eleni Panagopoulos, Ivelisse Page, Jennifer Durham, Michael Carducci, Channing Paller, Sushant Kachhap. Effect of mistletoe extract on growth and proliferation of prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB102.

An unconventional approach in the treatment of canine peritoneal mesothelioma with ultra-diluted Viscum album: Case report

Mesothelioma is a diffuse and primary neoplasm, arising from mesothelial cells that line the cavitary organs such as peritoneum, rarely found in dogs, it is aggressive and has a high mortality rate and no effective treatment with low therapeutic response to chemotherapy and radiotherapy: (1) Background: The objective of the clinical study was to use ultra-diluted Viscum album in monotherapy, analyzing the staging and anti-metastatic potential after splenectomy. The therapeutic methodology according to the Law of Similar described in Homeopathy was through the combination of ultra-diluted V.a in combined potencies. (2) Methods: The objective of the clinical study was to use ultra-diluted V.a in monotherapy, analyzing the staging and anti-metastatic potential after splenectomy. The therapeutic methodology according to the Law of similar described in Homeopathy was through the combination of ultra-diluted Viscum album in combined potencies by subcutaneous route. (3) Results: The results showed that the combined ultra-diluted Viscum album promoted modification of the cystic structures and staged the abdominal mass, preventing metastases for 30 months. (4) Conclusions: We conclude that although peritoneal mesothelioma is an extremely serious neoplasm, with high metastatic potential, the ultra-diluted Viscum album protocol increased survival without the use of any chemotherapeutic substance.

Determination of Phenolic Compounds, Organic Volatile Molecules and Anti-Cancer Properties in Inula Viscosa L., Viscum Album L. and Raphanus Sativus L.

The plants elecampane (Inula Viscosa L.), mistel (Viscum album L.) and black radish seed (Raphanus Sativus L.) have been used in the treatment of common diseases worldwide as part of traditional medicine for many years. Especially in Turkey, elecampane plant is commonly used as remedy of cancer. In this study, phytochemical components of these three plants were analyzed using liquid chromatography-mass spectrometer/mass spectrometer and gas chromatography-mass spectrometer techniques. Antioxidant activity of the characterized extracts were evaluated using DPPH assay, followed by biological properties were studied using MDA-MB-231 breast cancer line. Differences in the chemical compositions of the extracts resulted in alteration in antioxidant potentials, where elecampane gave the highest antioxidant activity while black radish seed extracts did not provide any meaningful results within the test period. Cytotoxicity studies showed that chemical composition is of the most prominent factor that defined the IC50 value of each extract, where pro-oxidant and antioxidant affects were observed in relation to presence of flavonoids. Mistel extract was further tested for wound healing and apoptosis tests, and the extract was obtained as a trigger for both apoptosis and wound-healing. The findings can be a basis for refinement as fractionation of the mistel and elecampane extracts so as to obtain the best mixture that can serve as strong anticancer agent mixture.

Metabolomic analysis of Viscum album L homeopathic tinctures and antitumor studies in 3D spheroid models

Background The antitumoral efficacy of aqueous Viscum album extracts is attributed to the presence of lectins and viscotoxins. However, previous studies demonstrated an antitumoral activity of European V. album ethanolic homeopathic tinctures (VAHT) prepared according to homeopathic methodology. Aims To investigate the seasonal influences (summer and winter) in the metabolomic profile of V. album ssp. homeopathic mother tinctures (VAHT) and to evaluate the antitumoral activity of some VAHT in 3D tumor spheroid models. Methodology The following VAHT were prepared by ethanolic maceration: V. album ssp. album growing on Malus domestica, Quercus sp. and Ulmus sp.; V. album ssp. austriacum from Pinus sylvestris; V. album ssp. abietis from Abies alba. Chemical analyses were performed using liquid chromatography coupled to high-resolution mass spectrometry. Data was submitted to multivariate statistical analysis using principal component analysis (PCA) and Partial Least Squares Discriminant Analysis (PLSDA) in Metaboanalyst platform. The antitumor potential of VAHT (0.5% v/v) was conducted in 3D tumor spheroid models (MDA-MB-231 cell line) by MTT for 72 hours. Results and discussion The PCA analysis explained 40% of data variation and clustered VAHT samples into 3 groups, emphasizing the chemical similarity between the botanical subspecies of V. album. Some key compounds were mainly responsible for this separation: pinobankasin hexose-pentose (V. album ssp. abietis); citric acid (V. album ssp. austriacum); malic acid (V. album ssp. album). The chemical differences among summer and winter samples, detected by PLSDA, were related to the Viscum album host trees. A significant reduction of 50% and 41% (p

Phytochemical Characterization of Phoradendron bollanum and Viscum album subs. austriacum as Mexican Mistletoe Plants with Antimicrobial Activity.

In Mexico, mistletoes have several applications in traditional medicine due to the great variety of compounds with biological activities that have not been characterized to date. The goals of the present study are to analyze the composition of minerals and phytochemical compounds in Mexican mistletoes Phoradendron bollanum and Viscum album subs. austriacum qualitatively and quantitatively, identify the compounds using HPLC-MS, and assess the antimicrobial potential in phytopathogenic microorganism control. Mineral content was evaluated with X-ray fluorescence. Three types of extracts were prepared: ethanol, water, and aqueous 150 mM sodium chloride solution. Characterization was carried out using qualitative tests for phytochemical compound groups, analytical methods for proteins, reducing sugars, total phenol, flavonoids quantification, and HPLC-MS for compound identification. The antimicrobial activity of mistletoe’s liquid extracts was evaluated by microplate assay. K and Ca minerals were observed in both mistletoes. A qualitative test demonstrated alkaloids, carbohydrates, saponins, flavonoids, tannins, and quinones. Ethanolic extract showed flavonoids, 3845 ± 69 and 3067 ± 17.2 mg QE/g for Phoradendron bollanum and Viscum album subs. austriacum, respectively, while aqueous extracts showed a total phenol content of 65 ± 6.9 and 90 ± 1.19 mg GAE/g Phoradendron bollanum and Viscum album subs. austriacum, respectively. HPLC-MS identified largely hydroxycinnamic acids and methoxycinnamic acids. Clavibacter michiganenses was successfully inhibited by aqueous extract of both mistletoes.

Impact of Harvest Conditions and Host Tree Species on Chemical Composition and Antioxidant Activity of Extracts from Viscum album L.

The content of plant secondary metabolites is not stable, and factors such as the region/location effect and seasonal variations have an impact on their chemical composition, especially in parasitic plants. Research in this area is an important step in the development of quality parameter standards of medicinal plants and their finished products. The effects of the time and place of harvest and the host tree species on the chemical composition and antioxidant activity of mistletoe extracts were investigated. Statistical tools were used to evaluate the results of the spectrophotometric and LC-ESI-MS/MS studies of the phenolic composition and antioxidant activity. The investigations indicate that the qualitative and quantitative composition, influencing the biological activity of mistletoe extracts, largely depends on the origin of the plant. The mistletoe extracts exhibited a rich phenol profile and high antioxidant activity. The chemometric analysis indicated that mistletoe collected from conifers (Viscum abietis and Viscum austriacum) had the most advantageous chemical composition and antioxidant activity. Moreover, the chemical profile and biological activity of the plant material were closely related to the climatic conditions and location of the harvested plant. Higher levels of phenolic compounds and high antioxidant activity were found in extracts obtained from plant material collected in cold weather with the presence of snow and less sunshine (autumn–winter period).

Squamous Cell Carcinoma in a Dog (Canis familiaris) Treated by the Viscum album Therapy – Case Report

Squamous Cell Carcinoma in a Dog (Canis familiaris) Treated by the Viscum album Therapy – Case Report

Comparative Study on Phytochemical Profile and Antioxidant Activity of an Epiphyte, Viscum album L. (White Berry Mistletoe), Derived from Different Host Trees.

The study aimed at evaluating the antioxidant profile of a medicinal epiphyte Viscum album L. harvested from three tree species, namely, Populus ciliata L, Ulmus villosa L., and Juglans regia L. The crude extracts were obtained with ethanol, methanol, and water and were evaluated for the total phenol content (TPC), total flavonoid content (TFC), and antioxidant activities using total reducing power (TRP), ferric reducing antioxidant power (FRAP), 1, 1-diphenyl 1-2-picryl-hydrazyl (DPPH), superoxide radical scavenging (SOR), and hydroxyl radical scavenging (•OH) assays. Our results showed that crude leaf extracts of plants harvested from the host Juglans regia L. exhibited higher yields of phytochemical constituents and noticeable antioxidative properties. The ethanolic leaf samples reported the highest phenols (13.46 ± 0.87 mg/g), flavonoids (2.38 ± 0.04 mg/g), FRAP (500.63 ± 12.58 μM Fe II/g DW), and DPPH (87.26% ± 0.30 mg/mL). Moreover, the highest values for TRP (4.24 ± 0.26 μg/mL), SOR (89.79% ± 0.73 mg/mL), and OH (67.16% ± 1.15 mg/mL) were obtained from aqueous leaf extracts. Further, Pearson correlation was used for quantifying the relationship between TPC, TFC, and antioxidant (FRAP, DPPH, SOR, OH) activities in Viscum album L. compared to their hosts. It was revealed that the epiphyte showed variation with the type of host plant and extracting solvent.