Viscumalbum L. homeopathic mother tinctures: metabolome and antitumor activity

Viscum album L. is a semi-parasitic plant with antitumor activity attributed to theaqueous extracts. However, European V. album ethanolic extracts (VAE) have also demonstrated invitro activity in tumor models. Aims: Evaluate the metabolic profiles of fifty VAE harvested duringsummer and winter seasons and their antitumor activity through 2D and 3D models. Methodology:VAEwerepreparedbymacerationfrom:V.albumsubsp.albumgrowingonMalus domestica,Quercus sp.and Ulmus sp.; V. album subsp. austriacum from Pinus sylvestris; V. album subsp. abietis from Abies alba.Chemical analyses were performed through liquid chromatography coupled with high resolutionmass spectrometry and Partial Least Squares Discriminant Analysis (PLS-DA) was performed in theMetaboanalyst 4.0. The antitumor potential of the selected VAE was evaluated in 2D and 3D models(MDA-MB-231 cancer cells) by MTT, crystal violet and glycolytic pathway analysis. Results anddiscussion:Thefirst3principalcomponentsinPLS-DAexplained60%and40%ofdatavariationin positive andnegativemodesrespectively.Threegroupswereformedandshowedchemicalsimilarityamong V. album subspecies. The compounds responsible for group separation were tentativelyidentifiedas:pinobankasinornaringenin hexoside;isorhamnetin-3-hexoside,meglutolanddifferent aminoacids.ThesummerVAEat0.5%v/vinducedhighercytotoxicdamagethanthewinterpreparations, and Abies alba and Quercus sp. VAE promoted 49% and 42% reduction of tumorviability in 3D model (72h incubation), respectively. MDA-MB-231 glycolytic pathway in 2D modelshowed a decrease in the glucose consumption and extracellular lactate production. Also, PFK (6-phosphofructo-1-kinase)andPK(Pyruvatekinase)activitieswereinhibitedbyAbiesalbaandQuercus sp. VAE at 48h of incubation. Conclusion: VAE extracts showed different metabolomes andthe glycolytic pathway should be an important target involved in the inhibition of tumor growth bytheseextracts

Series of experiments performed with the droplet evaporation method on low potencies

Homeopathic preparations in low potencies, containing still measurable quantities of the starting substance, constitute a unique research field in homeopathic basic research. Here a series of experiments is presented carried out by means of the droplet evaporation method (DEM), investigating the specificity of the method, and presumed effects of the succussion procedure applied in the production of homeopathic preparations. Methods:DEM analysis consisted in the evaporation of droplets of the potencies perse placed on microscope slides. Resulting patterns were photographed. Images were evaluated by means of ImageJ software, by measuring grey level distribution, texture, and fractality. The experimentation consisted of four series: (i) screening (1x­6x potencies from 19 substances), (ii) differentiation experiments (2x­6x potencies of Echinacea, Baptisia, Luffa, and Spongia), (iii) differentiation between succussed (100 or 10 times) and unsuccussed samples (Echinacea 2x, Baptisia 3x, Baptisia 4x, Luffa 4x, and Spongia 6x). (iv) investigation of the influence upon the patterns of single compounds present in a remedy complex. The experimental set-up stability was examined by systematic positive control experiments. Results:(i) Homeopathic preparations of mineral origin showed the greatest form variety, whereas those of vegetal origin created fractal patterns in the potency range 2x­4x. (ii) Differentiation of potencies of different origin at the same dilution level was possible from 2x to 4x. (iii) In all potency levels, succussed (100 and 10 times) and unsuccussed variants could be significantly differentiated. Significant differences between all variants were found in some cases in potency levels 4x and higher. In general, application of succussion reduced size, homogeneity, and complexity of the DEM patterns. (iv) Patterns of a remedy complex Luffa 4x -Mercurius bijodatum 9x showed a clear predominance of the Luffa 4x; however also the second component, present in a much lower concentration, influenced significantly the pattern of the remedy complex as also differed significantly from the pattern of succussed water control. Conclusions:The results suggest that DEM is a suitable tool for scientific investigation of homeopathic preparations in the low potency range. DEM might be applied to assess further research questions, such different potentization procedures (vessel shape, overhead volume, material), storing time, and difference between batches.

In vitro evaluation of the effects of Viscum album homeopathic preparations on murine melanoma cells

Viscum album extract (VA) is a complementary treatment in cancer, with in vitro and in vivo cytotoxic effects on several tumor types when applied in phytochemical doses. However, highly diluted ethanolic homeopathic preparations' effects and mechanisms need further study. Aims:To assess the in vitro effects of highly diluted VA from the subspecies V. album abietis and V. album album at different potency levels in different dilution ratios on murine melanoma cells. Methodology:The VA mother tinctures (MT)from Abies alba (MTA) and Quercus robur (MTQ) were prepared with summer and winter samples, harvested in Switzerland. They were submitted to homeopathic ethanolic maceration and a subsequent dynamization process. MTA, MTQ and the following respective potencies were tested in B16F10 murine cells: 3x, 12x, 30x, 6cH, 12cH, 200cH, 2LM, 3LM, and 5LM. Dynamized water, dynamized and non-dynamized ethanol, and carboplatin were used as control groups. The mitochondrial activity and cell viability analysis were performed at 1, 24, 48, and 72 hours by in vitro incubation. MTA and MTQ harvested in summer, as well as 12x, 200cH and 5LM potencies were also tested to cell apoptosis and necrosis markers, reactive oxygens species (ROS) production, inflammatory cytokines profile, cell morphology, and migratory capacity. Results and discussion: MTA and MTQ induced a decrease in cell metabolism and higher cytotoxicity within 1 hour, with significant morphological changes and increased production of ROS and inflammatory cytokines. Both homeopathic dilutions 12x and 5LM showed an influence on cell metabolism, cell replication, and oxidative stress modulation with inflammatory cytokines, mitosis, and migration pattern changes. On the other hand, Quercus robur and Abies alba 200cH showed increased on cytotoxicity and ROS levels, respectively. Conclusion:The in vitro effects of Viscum album homeopathic solutions in melanoma cells highlight the promising antitumoral potential and reinforce the need for further research to better understanding their mechanisms of action.

Formation of Self-organized Structures in Drying Droplets: a Promising Analytical Tool for Homeopathy Basic Research

The droplet evaporation method (DEM) is based on the evaporation-induced pattern formation in droplets and is applied mainly for medical diagnosis[1].Here, we present aseries of experiments performed by our team showing DEMs potential also forhomeopathy basic research, in particular, for the investigation of(i) low potencies, (ii) low potency complexes (physical model), and (iii) the action of high potencies (plant-based model).Methods:(i) DEM differentiated significantly between Luffa, Baptisia, Echinacea, and Spongiauntil 4x[2]. Furthermore, the patterns varied in function of the numberof succussion strokes (0, 10, or 100) applied during potentization[3]. The performance of chaotic succussions vs. laminar flow vs. slight mixing during the potentization of Viscum album quercus3x influenced the DEM patterns; the chaotic succussions reduced, whereas laminar flow enhanced the patterns complexity vs. the unsuccussed control.(ii) The addition of Mercurius bijodatus9x to Luffa4x changed significantly the DEM patterns, even if the material quantity present in the 9x potency lied far beyond that of ultrapure water.(iii) Leakages obtained by placing healthy or arsenic-damaged wheat-seeds into Arsenicum album45x orheat-damaged intoZincum metallicum30c vs. water created significantly different DEM structures [4, 5]. Results:The damaged seeds put into the potency created structures characterized by a higher complexity than those obtained from damaged seeds put into control water. Furthermore, the potency action seemed to increase with rising numbers ofsuccussion strokes applied during potentization,ascould be shown by means of DEM patterns and germination rate using the same wheat-seed model[6].In all our studies, the pattern evaluation was computerized (texture and fractal analysis performed by means of ImageJ) or based on deep-learning algorithms and the robustness of the experimental system was checked by means of systematic control experiments.Conclusion:DEM together with other similarmethods has also been reviewed by our team for what concerns theapplication in homeopathy basic research[7].

Pesquisas físico-químicas sobre as preparações homeopáticas: revisão sistemática e análise bibliométrica ­ parte 1 / Physical-chemical research on homeopathic preparations: review systematics and bibliometric analysis

Objetivo: A última revisão sistemática das pesquisas físico-químicas sobre preparações homeopáticas foi publicada em 2003. O objetivo do presente estudo foi atualizar e ampliar o estado atual dos conhecimentos na área das propriedades físico-químicas das preparações homeopáticas. Na parte 1 do estudo objetivamos apresentar um panorama da literatura quanto à qualidade e métodos utilizados nas publicações. Na parte 2 objetivamos identificar as técnicas experimentais mais interessantes. Desta maneira, esperamos estar na posição de gerar hipóteses significativas quanto ao possível modo de ação das preparações homeopáticas. Métodos: Foi adotado um procedimento com 2 etapas: 1) pesquisa ampla da literatura, seguida de análise bibliométrica e da qualidade das publicações e, 2) análise qualitativa minuciosa das pesquisas físico-químicas individuais localizadas. Na presente publicação relatamos a etapa 1). Realizamos uma busca nas principais bases científicas de dados de publicações relatando pesquisas físico-químicas da homeopatia desde a sua origem até o final de 2015. As publicações foram avaliadas através de um sistema de pontuação, o Manuscript Information Score (MIS). Foram coletadas informações sobre o país de origem dos estudos e as técnicas experimentais utilizadas. Resultados: Localizamos 183 publicações (versus 44 na revisão anterior), 122 delas com MIS ≥ 5. A taxa de publicações na área foi ˜2 por ano entre as década de 1970 e 2000. Depois aumentou para mais de 5,5 publicações por ano. [...] Conclusões: As pesquisas físico-químicas sobre as preparações homeopáticas está aumentando tanto na quantidade e na qualidade das publicações. (AU)

Objectives: The last systematic review of physicochemical research performed on homeopathic preparations was published in 2003. The aim of the study is to update and expand the current state of knowledge in the area of physicochemical properties of homeopathic preparations. In part 1 of the study, we aim to present an overview of the literature with respect to publication quality and methods used. In part 2, we aim to identify the most interesting experimental techniques. With this, we aim to be in a position to generate meaningful hypotheses regarding a possible mode of action of homeopathic preparations. Methods: A two-step procedure was adopted: (1) an extensive literature search, followed by a bibliometric and quality analysis on the level of publications and (2) a thorough qualitative analysis of the individual physicochemical investigations found. In this publication, we report on step (1). We searched major scientific databases to find publications reporting physicochemical investigations of homeopathy from its origin to the end of 2015. Publications were assessed using a scoring scheme, the Manuscript Information Score (MIS). Information regarding country of origin of the research and experimental techniques used was extracted. Results: We identified 183 publications (compared to 44 in the last review), 122 of which had an MIS ‡5. The rate of publication in the field was *2 per year from the 1970s until 2000. Afterward, it increased to over 5.5 publications per year. [...]Conclusions: Physicochemical research into homeopathic preparations is increasing both in terms of quantity and quality of the publications. (AU)

Pesquisas físico-químicas sobre as preparações homeopáticas: revisão sistemática e análise bibliométrica ­ parte 1 / Physical-chemical research on homeopathic preparations: review systematics and bibliometric analysis

Objetivo: A última revisão sistemática das pesquisas físico-químicas sobre preparações homeopáticas foi publicada em 2003. O objetivo do presente estudo foi atualizar e ampliar o estado atual dos conhecimentos na área das propriedades físico-químicas das preparações homeopáticas. Na parte 1 do estudo objetivamos apresentar um panorama da literatura quanto à qualidade e métodos utilizados nas publicações. Na parte 2 objetivamos identificar as técnicas experimentais mais interessantes. Desta maneira, esperamos estar na posição de gerar hipóteses significativas quanto ao possível modo de ação das preparações homeopáticas. Métodos: Foi adotado um procedimento com 2 etapas: 1) pesquisa ampla da literatura, seguida de análise bibliométrica e da qualidade das publicações e, 2) análise qualitativa minuciosa das pesquisas físico-químicas individuais localizadas. Na presente publicação relatamos a etapa 1). Realizamos uma busca nas principais bases científicas de dados de publicações relatando pesquisas físico-químicas da homeopatia desde a sua origem até o final de 2015. As publicações foram avaliadas através de um sistema de pontuação, o Manuscript Information Score (MIS). Foram coletadas informações sobre o país de origem dos estudos e as técnicas experimentais utilizadas. Resultados: Localizamos 183 publicações (versus 44 na revisão anterior), 122 delas com MIS ≥ 5. A taxa de publicações na área foi ˜2 por ano entre as década de 1970 e 2000. Depois aumentou para mais de 5,5 publicações por ano. [...] Conclusões: As pesquisas físico-químicas sobre as preparações homeopáticas está aumentando tanto na quantidade e na qualidade das publicações. (AU)

Objectives: The last systematic review of physicochemical research performed on homeopathic preparations was published in 2003. The aim of the study is to update and expand the current state of knowledge in the area of physicochemical properties of homeopathic preparations. In part 1 of the study, we aim to present an overview of the literature with respect to publication quality and methods used. In part 2, we aim to identify the most interesting experimental techniques. With this, we aim to be in a position to generate meaningful hypotheses regarding a possible mode of action of homeopathic preparations. Methods: A two-step procedure was adopted: (1) an extensive literature search, followed by a bibliometric and quality analysis on the level of publications and (2) a thorough qualitative analysis of the individual physicochemical investigations found. In this publication, we report on step (1). We searched major scientific databases to find publications reporting physicochemical investigations of homeopathy from its origin to the end of 2015. Publications were assessed using a scoring scheme, the Manuscript Information Score (MIS). Information regarding country of origin of the research and experimental techniques used was extracted. Results: We identified 183 publications (compared to 44 in the last review), 122 of which had an MIS ‡5. The rate of publication in the field was *2 per year from the 1970s until 2000. Afterward, it increased to over 5.5 publications per year. [...]Conclusions: Physicochemical research into homeopathic preparations is increasing both in terms of quantity and quality of the publications. (AU)