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Twelve bufadienolides and six 19-norbufadienolides were isolated from the aerial parts of Helleborus foetidus. They consist of aglycons and glucosides and include nine previously undescribed compounds and a compound reported for the first time as a genuine natural product. Their structures were established by extensive spectroscopic analysis and the structure and absolute configuration of two previously unreported 3,4-epoxy derivatives were confirmed by single crystal X-ray diffraction analysis. The compounds were tested for their cytotoxicity on MCF-7 human breast cancer cells. They show differential cytotoxic activity with IC 50 values in the range of 2.4 nM - >10 μM. The potency of the activity strongly correlates with the presence of a C-19 aldehyde group. The data complement the scientific basis underpinning the use of H. foetidus in anthroposophic medicine for the integrative treatment of cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Pharmacopoeias regulate the manufacture of potentised pharmaceutical preparations used in different branches of complementary and integrative medicine. The physicochemical properties and biological activity of these preparations are often investigated in preclinical research, yet no guidelines for experimental research currently exist in this area. The present PrePoP guidelines aim to provide recommendations to promote high-quality, statistically sound, and reproducible preclinical research on potentised preparations., Methods: Input was gathered from researchers nominated by the relevant scientific societies using a simplified Delphi consensus approach covering the most relevant aspects of basic research methodology in the field including appropriate controls, sample preparation and handling, and statistics. After three rounds of feedback, a consensus was finally reached on the most important aspects and considerations for conducting high-quality research on potentised preparations., Results: We present a series of recommendations on a range of topics including experimental controls, system stability, blinding and randomisation, environmental influences, and procedures for the preparation of potentised samples and controls, and we address some specific challenges of this research field., Conclusion: This expert consensus process resulted in a robust set of methodological guidelines for research on potentised preparations and provides a valuable framework that will inform and improve the quality of subsequent research in this emerging field., Please Cite This Article as: Tournier AL, Bonamin LV, Buchheim-Schmidt S, Cartwright S, Dombrowsky C, Doesburg P, Holandino C, Kokornaczyk MO, van de Kraats EB, López-Carvallo JA, Nandy P, Mazón-Suástegui JM, Mirzajani F, Poitevin B, Scherr C, Thieves K, Würtenberger S, Baumgartner S. Scientific guidelines for preclinical research on potentised preparations manufactured according to current pharmacopoeias-the PrePoP guidelines. J Integr Med. 2024; 22(5): 533-544., (Copyright © 2024 Shanghai Yueyang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine. Published by Elsevier B.V. All rights reserved.)

Background and Purpose: Natural products are potential sources of anticancer components. Among various species, the lipophilic extract of the Viscum album subsp. austriacum (Wiesb.) Vollm. (VALE) has shown promising therapeutic potential. The present work aimed to qualify the plant source and characterize the extract's chemical profile. In addition, a self-nanoemulsifying drug delivery system (SNEDDS) containing VALE (SNEDDS-VALE) was developed., Methods: V. album subsp. austriacum histochemistry was performed, and the chemical profile of VALE was analyzed by GC-MS. After the SNEEDS-VALE development, its morphology was visualized by transmission electron microscopy (TEM), while its stability was evaluated by the average droplet size, polydispersity index (PdI) and pH. Lastly, SNEDDS-VALE chemical stability was evaluated by LC-DAD-MS., Results: The histochemical analysis showed the presence of lipophilic compounds in the leaves and stems. The major compound in the VALE was oleanolic acid, followed by lupeol acetate and ursolic acid. SNEDDS was composed of medium chain triglyceride and Kolliphor ® RH 40 (PEG-40 hydrogenated castor oil). A homogeneous, isotropic and stable nanoemulsion was obtained, with an average size of 36.87 ± 1.04 nm and PdI of 0.14 ± 0.02, for 14 weeks., Conclusion: This is the first histochemistry analysis of V. album subsp. austriacum growing on Pinus sylvestris L . which provided detailed information regarding its lipophilic compounds. A homogeneous, isotropic and stable SNEDDS-VALE was obtained to improve the low water solubility of VALE. Further, in vitro and in vivo experiments should be performed, in order to evaluate the antitumoral potential of SNEDDS-VALE., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Pereira et al.)

Background:  Previously we developed a test system which yielded highly significant evidence for specific effects of a Stannum metallicum 30x preparation in a multi-center replication trial. This test system is based on cress seed germination in homeopathic or control samples, CuCl 2 crystallization of the cress extract, and subsequent digital textural image analysis of the resulting crystallization patterns., Objectives:  The current study aimed to investigate whether three novel outcome parameters could further corroborate and possibly characterize the specific effects of Stannum metallicum 30x., Methods:  To this end, (1) cress seedling length, (2) a second texture analysis parameter, entropy and (3) the local connected fractal dimension (LCFD) of crystallization patterns as a measure of complexity were considered. The stability of the experimental setup was monitored throughout the entire investigation with systematic negative control (SNC) experiments., Results:  Cress length and entropy revealed a time-modulated potency treatment effect, in the absence of a significant main treatment effect. This indicated that the effect of the potency treatment varied significantly across the different experimental days. LCFD yielded a highly significant potency treatment effect. In addition, a significant interaction of treatment with experimental day seems to indicate a modulation of this effect. No significant effects were observed in any of the evaluations of the SNC experiments, indicative of a stable experimental setup and a reliable and specific treatment effect. Neither significant nor strong correlations were found between the four parameters, indicating that they reflect different effects of Stannum metallicum 30x on the organism treated., Conclusion:  This quadruple characterization of the biological effects of Stannum metallicum 30x provides an unprecedented opportunity for basic homeopathy research into, among others, the presumed specificity of homeopathic preparations., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Various types of motion introduced into a solution can affect, among other factors, the alignment and positioning of molecules, the agglomeration of large molecules, oxidation processes, and the production of microparticles and microbubbles. We employed turbulent mixing vs. laminar flow induced by a vortex vs. diffusion-based mixing during the production of Viscum album Quercus L. 10 -3 following the guidelines for manufacturing homeopathic preparations. The differently mixed preparation variants were analyzed using the droplet evaporation method. The crystalline structures formed in dried droplets were photographed and analyzed using computer-supported image analysis and deep learning. Computer-supported evaluation and deep learning revealed that the patterns of the variant succussed under turbulence are characterized by lower complexity, whereas those obtained from the vortex-mixed variant are characterized by greater complexity compared to the diffusion-based mixed control variant. The droplet evaporation method could provide a relatively inexpensive means of testing the effects of liquid flow and serve as an alternative to currently used methods., (© 2024. The Author(s).)

Introduction: European mistletoe ( Viscum album L.) has been gaining increasing interest in the field of oncology as a clinically relevant adjunctive treatment in many forms of cancer. In the field of phytopharmacology, harvesting time is pivotal. In the last century, a form of metabolomic fingerprinting based on pattern formation was proposed as a way to determine optimal harvesting times to ensure high quality of mistletoe as raw material for pharmaceutical use. In order to further evaluate the information obtained with this metabolomic fingerprinting method, we analysed a large time series of previously undigitised daily mistletoe chromatograms dating back to the 1950s. Methods: These chromatograms were scanned and evaluated using computerized image analysis, resulting in 12 descriptors for each individual chromatogram. We performed a statistical analysis of the data obtained, investigating statistical distributions, cross-correlations and time self-correlations. Results: The analysed dataset spanning about 27 years, contains 19,037 evaluable chromatograms in daily resolution. Based on the distribution and cross-correlation analyses, the 12 descriptors could be clustered into six independent groups describing different aspects of the chromatograms. One descriptor was found to mirror the annual rhythm being well correlated with temperature and a phase shift of 10 days. The time self-correlation analysis showed that most other descriptors had a characteristic self-correlation of ∼50 days, which points to further infradian rhythms (i.e., more than 24 h). Discussion: To our knowledge, this dataset is the largest of its type. The combination of this form of metabolomic fingerprinting with the proposed computer analysis seems to be a promising tool to characterise biological variations of mistletoe. Additional research is underway to further analyse the different rhythms present in this dataset., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Guglielmetti, Baumgartner, Scherr, Martin and Tournier.)

Introduction: Larch oleoresin has been described regarding several biological activities and medicinal applications, such as wound healing and treatment of ulcers, but little is known about its chemical composition., Material and Methods: Eight oleoresins from Larix decidua Mill. obtained from four companies and one adulterated control were therefore investigated to determine their content of essential oils and to verify possible differences in their composition in relation to the harvest and manufacturing processes. Essential oils (EOs) were isolated by distillation and the yield was analysed., Results and Discussion: The yield of EO varied among all samples. The yield of the pure larch samples covered a range of 7.8% to 15.5%. A higher yield (19.0%) was observed for adulterated control, which contained oleoresins from different Pinaceae trees. Age of samples had no impact on yield. However, there was a significant statistical variation (p<0.05) in the yields of the mid-summer oleoresins (>10%) compared to early or late summer (<10%), emphasising the importance of the time of collection. Samples were subsequently analysed by GC-MS. EO samples confirmed the presence of various chemical classes, such as monoterpenes, sesquiterpenes, and diterpenes. α-pinene was the compound with the highest concentrations (>50%), followed by β-pinene (>6%), D-limonene (>2.5%), α-terpineol (>0.9%), β-myrcene (>0.2%), and 3-carene (>0.05%). Samples were grouped using multivariate data analysis (MVDA) with respect to the chemical variation between the oleoresins' EOs. The resulting four clusters were named low (low yield obtained for the samples), mixed (mixed oleoresin from different Pinaceae species, adulteration control), old (old oleoresin kept in the institute), and normal (other oleoresins) samples, each presenting distinct chemical biomarkers. There were considerable differences between site and time of collection. Essential oil yield did not always meet requirements as defined by the German Homeopathic Pharmacopoeia. In addition, adulterated or aged samples could be identified as compared to pure and fresh larch oleoresins., Conclusion: We conclude that larch oleoresin used for pharmaceutical applications has to be carefully analysed and standardised to guarantee reproducible product quality., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Batista, Melo, Holandino, Maier, Huwyler, Baumgartner and Boylan.)

Background: Few measurements of fatigue and quality of life have been performed during neoadjuvant chemotherapy of early breast cancer. This study evaluates fatigue and quality of life experienced by early breast cancer patients during neoadjuvant chemotherapy and their association with different clinical parameters., Methods: Fifty-four stage I-III patients' responses to the Multidimensional Fatigue Inventory (MFI) and to the Functional Assessment of Cancer Therapy-Breast (FACT-B) were analyzed by a linear covariance pattern model. Chemotherapy regimen, age, baseline fatigue level, body-mass-index and cancer stage were added to the model to estimate their impact on both outcomes., Results: All fatigue dimensions worsened in clinically relevant levels. Physical fatigue worsened the most, mental fatigue the least. For quality of life, physical and functional well-being worsened the most. Only emotional well-being improved during chemotherapy. Physical well-being worsened more during standard than during dose-dense chemotherapy, and more during anthracycline than during taxane cycles. Age, body-mass-index and cancer stage had no impact. The higher the fatigue levels at baseline, the less they worsened during chemotherapy., Conclusions: Further actions to reduce fatigue and improve quality of life during neoadjuvant chemotherapy of early breast cancer are needed. Focus should be laid on the physical dimension. Future research should also investigate the impact of different chemotherapy sequences and densities on fatigue and quality of life., Study Registration: The study was registered in the German Clinical Trials Register in May 2019 (DRKS00016761)., (© 2023. The Author(s).)

Introduction: Cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC) belong to the group of keratinocyte carcinomas (KC). Actinic keratosis (AK) is a precursor lesion of cSCC. The incidences of cSCC, BCC, and AK are currently strongly increasing. Different standard therapies exist for these conditions but are not always applicable or successful. Hydrophilic Viscum album extracts have been used in anthroposophic cancer therapy since 1917. Viscum album lipophilic extract (VALE) is prepared by means of supercritical CO2 extraction. This retrospective case series assessed the safety and clinical effects of a topical application of 10% VALE in individual cases of cSCC, BCC, and AK., Methods: For this retrospective case series, a positive vote was obtained from the Ethics Committee of the University of Witten/Herdecke (No. 146/2020). Eligible patients signed a declaration of consent prior to inclusion in the study. The main outcome parameters were the clinical response to treatment with VALE and adverse drug reactions. Risk factors, concomitant therapies and diseases, further diagnostic and therapeutic information were documented where available. Data analysis was performed on the level of patients and of individual lesions., Results: The study population consisted of 55 patients with 74 skin lesions. Individual case analysis accompanied by photographic documentation revealed typical and promising treatment courses. Clinical response rates (complete + partial remissions) for individual lesions were 78% for cSCC, 70% for BCC, and 71% for AK. Complete remission rates for individual lesions were 56% for cSCC, 35% for BCC, and 15% for AK. In cSCC and BCC, shorter times to best clinical response were observed. Adverse drug reactions were reported in 5 patients including erythema and inflammatory reactions of mostly moderate severity that resolved completely. In one case, therapy was temporarily paused, in four cases it was continued without interruption., Discussion/conclusion: The results of this study suggest that VALE is a safe and tolerable extract under whose application complete and partial remissions of KC could be observed. To improve and assess the efficacy of VALE, prospective investigations are necessary., Das kutane Plattenepithelkarzinom (cSCC) und das Basalzellkarzinom (BCC) gehören zur Gruppe der Keratinozytenkarzinome (KC). Die aktinische Keratose (AK) ist eine Vorstufe des cSCC. Die Häufigkeit von cSCC, BCC und AK ist derzeit stark im Steigen begriffen. Es gibt verschiedene Standardtherapien für diese Erkrankungen, die jedoch nicht immer anwendbar oder erfolgreich sind. Hydrophile Viscum album-Extrakte werden seit 1917 in der anthroposophischen Krebstherapie eingesetzt. Viscum album Lipophilic Extract (VALE) wird mittels überkritischer CO2-Extraktion hergestellt. In dieser retrospektiven Fallserie wurden die Sicherheit und die klinischen Auswirkungen einer topischen Anwendung von 10% VALE bei einzelnen Fällen von cSCC, BCC und AK untersucht.Für diese retrospektive Fallserie wurde ein positives Votum der Ethikkommission der Universität Witten/Herdecke eingeholt (Nr. 146/2020). Die in Frage kommenden Patienten unterzeichneten vor der Aufnahme in die Studie eine Einverständniserklärung. Die wichtigsten Ergebnisparameter waren das klinische Ansprechen auf die Behandlung mit VALE und unerwünschte Arzneimittelwirkungen. Risikofaktoren, Begleittherapien und -erkrankungen sowie weitere diagnostische und therapeutische Informationen wurden, soweit vorhanden, dokumentiert. Die Datenanalyse wurde für jeden einzelnen Patienten und jeder einzelnen Läsion durchgeführt.Die Studienpopulation bestand aus 55 Patienten mit 74 Hautläsionen. Die Einzelfallanalyse mit Fotodokumentation ergab typische und vielversprechende Behandlungsverläufe. Die klinischen Ansprechraten (vollständige + partielle Remissionen) für einzelne Läsionen lagen bei 78% für cSCC, 70% für BCC und 71% für AK. Die Komplettremissionsraten für einzelne Läsionen lagen bei 56% für cSCC, 35% für BCC und 15% für AK. Bei cSCC und BCC wurden kürzere Zeiten bis zum besten klinischen Ansprechen beobachtet. Bei fünf Patienten wurden unerwünschte Arzneimittelwirkungen gemeldet, darunter Erytheme und Entzündungsreaktionen von meist mäßigem Schweregrad, die vollständig abklangen. In einem Fall wurde die Therapie vorübergehend pausiert, in vier Fällen wurde sie ohne Unterbrechung fortgesetzt.Die Ergebnisse dieser Studie deuten darauf hin, dass VALE ein sicherer und verträglicher Extrakt ist, bei dessen Anwendung vollständige und partielle Remissionen von KC beobachtet werden konnten. Um die Wirksamkeit der Therapie mit VALE weiterzuentwickeln und eingehender zu beurteilen, sind prospektive Untersuchungen erforderlich., (© 2024 S. Karger AG, Basel.)

Background: Mainly due to the use of different inclusion criteria and quality assessments, systematic reviews (SRs) and meta-analyses (MAs) with homeopathic intervention studies (HOMIS) have shown inconsistent results. We aimed to build recommendations for "Summarizing evidence from Homeopathic Intervention Studies" (Sum-HomIS recommendations) in order to approach standardization., Methods: Against the background of a framework-project to update the evidence from homeopathic intervention studies, we launched an expert panel on how to assess the quality of HOMIS and how to summarize evidence from HOMIS. The results of a literature review and the expert communications in advance of the panel as well as the consensus from the discussions are presented here. We added specific considerations for homeopathic veterinary research., Results: On top of the general guidelines when planning a review we report five basic Sum-HomIS recommendations. These are: 1) A broad literature search including special archives and consideration of so-called grey-literature; 2) The inclusion of controlled observational studies alongside randomized controlled trials; 3) The choice of a clear clinical research question in the terms that, if possible, the review project includes studies with predominantly homogeneous populations, interventions, comparators and outcomes (PICOs); 4) The use of a global quality assessment including the assessment of external, model and internal validity; 5) A summary of evidence using the GRADE-approach if the body of evidence is sufficiently large and homogenous or a descriptive summary if it is not so., Conclusions: We present recommendations for designing, conducting, and reporting SRs and MAs with HOMIS., Competing Interests: Declaration of Competing Interest Authors declare none., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Homeopathy is the subject of frequent debates, especially in public media. This systematic review aims to give an overview of conceptual criticisms of homeopathy in the scientific literature. The literature search was conducted in four databases (EMBASE, PubMed, Web of Science, PhilPapers) on August 25, 2020. Included were peer reviewed articles in English or German criticising the basic concepts of homeopathy as main topic; excluded were articles criticising homeopathy primarily based on analysis of empirical clinical and/or preclinical data. The formal structure of publications included was evaluated regarding the recommended structure for scientific publications (IMRaD, acronym for 'Introduction', 'Methods', 'Results' and 'Discussion'). Arguments criticising the concepts of homeopathy were extracted and classified into groups. The literature search revealed 5139 articles, of which 15 articles (published between 1959 and 2020) met the inclusion criteria. These articles complied only partly with the IMRaD structure; just four articles considered with 8 or 9 IMRaD criteria the majority of the defined 11 IMRaD criteria. Extracted arguments against the concepts of homeopathy were classified into five groups: 'Conflict with current scientific principles and the foundations of modern medicine', 'Lack of a scientific basis', 'Arguments based on scientific theories', 'Ethical considerations and social consequences', 'Lack of empirical clinical evidence'. This classification is intended to provide a basis for future in-depth scientific analyses and discussions. Based on the number of articles found in the peer reviewed literature, it can be concluded that the on-going discussion about homeopathy in the public media is not reflected in a corresponding academic debate., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Background: Homeopathic complex remedies, composed of several homeopathic medicines in the low potency range, are frequently used in the treatment of a number of common disorders. At the same time, they represent an almost unexplored area of research. Are complex remedies just additive mixtures of the components, or are there interactions between the latter leading to new properties of the complex?, Methods: In the present study, we analyzed as an example the simple bi-component complex, Luffa 4x - Mercurius bijodatus 9x, by means of patterns from evaporated droplets and tested what influences the complex's single compounds have upon the patterns and if there are any interactions. For this purpose, we compared in a series of five experiments patterns from evaporated droplets of the complex, Luffa 4x - Mercurius bijodatus 9x, and three comparison samples in which one or both of the complex's compounds were replaced by potentized solute. The patterns were photographed and evaluated for their gray-level distribution and texture using the software ImageJ . The experimental set-up's stability was tested by means of systematic control experiments., Results: We found that Mercurius bijodatus 9x significantly influenced the patterns of Luffa 4x, increasing their homogeneity; at the same time, the patterns of Mercurius bijodatus 9x combined with solvent were more heterogeneous than those obtained from a control consisting of two pure solvents., Conclusion: In this phenomenological assay, the complex Luffa 4x - Mercurius bijodatus 9x does not correspond to a simple addition of the components. The exact nature of the underlying interaction needs to be elucidated in further investigations., Competing Interests: None declared., (The Faculty of Homeopathy. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

The genus Viscum comprises a large number of semi-parasitic shrubs popularly known as Mistletoe. The Viscum species grow in many countries of Europe, Africa and Asia with different popular uses in ornamentation, foods and medicine. Many studies about Viscum have been done over the last years focusing on biological activities and chemical composition of the aqueous extracts, mainly related to anthroposophical medicines. However, it is known that non-aqueous preparations, as alcoholic extracts, have demonstrated different biological activities that are species-and host tree-dependent. Considering the potential of these alcoholic extracts, a scoping review was conducted using data from three online databases: PubMed, Scopus and Embase. Inclusion criteria consisted of the in vitro, in vivo, ex vivo, clinical and chemical studies of alcoholic extracts from Viscum species. The present review summarized 124 original publications about fourteen Viscum species. Viscum album , Viscum articulatum and Viscum coloratum were the main studied species. Alcoholic extracts demonstrated hypotensive, anticancer, antimicrobial, analgesic and anti-inflammatory capabilities, among other biological activities. Flavonoids, phenolic acids and terpenoids represented 48%, 24% and 11% of the total identified compounds, respectively. This review contributes to the knowledge of alcoholic preparations of the Viscum species and points out the lack of clinical studies concerning these different extracts.

Viscum album L. (Santalaceae) is an important medicinal plant traditionally used to treat several diseases, including cancer therapy. This paper provides detailed morpho-anatomical characteristics of the leaves, stems and berries of Viscum album subsp. album growing as hemi-parasite on the branches of Malus domestica (Suckow) Borkh. (Rosaceae) to aid species identification and botanical characterization. Additionally, for the first time, microchemical analyses of all tissues and Energy Dispersive X-Ray Spectroscopy analyses of the calcium oxalate crystals are provided for the first time. The plant features leathery presents green leaves with parallel veins, small yellow unisexual flowers in 3-flowered cymes, and the dioecious inflorescences usually consist of three flowers, with female flowers generating white fleshy berries, in which a seed is embedded in the mucilaginous mesocarp, normally containing two embryos. Anatomically, the analyzed leaves were isobilateral and amphistomatic, and showed straight anticlinal epidermal cell walls, thick cuticles with epicuticular wax crystalloids, and paracytic stomata. The midrib is flat on both sides and has a single vascular bundle, whereas the strongly shortened petiole is concave-convex in shape and contains five bundles. The stems show a primary structure with a ring of nine vascular bundles enclosing the pith. Calcium oxalate druses and cubic and quadrangular prisms were observed in different plant parts. The results of this study provide new microscopy information that can help in the authentication of mistletoe raw materials., (© 2023. The Author(s).)

Introduction: The introspective experience of cancer patients using mistletoe therapy has received little scientific interest, although it is crucial for a holistic understanding of this therapy. This study contributes to patient-centered research and treatment by documenting the subjective experiences of individuals undergoing mistletoe therapy., Methods: In this qualitative, explorative study, 20 outpatients with a history of various cancer types were recruited from Arlesheim Hospital (Arlesheim, Switzerland). All patients received subcutaneous mistletoe therapy for at least 2 years (median 7.5 years). Data was collected through 2 semi-structured, in-depth interviews per patient. Qualitative content analysis was applied to examine the data. The individual experience of mistletoe therapy was analyzed in relation to 6 predefined levels of human experience: physical, vital, emotional, mental, spiritual and social. In addition, 3 further aspects, considered as cross-dimensional perspectives, emerged out of the material: warmth, immune strengthening, and general wellbeing., Results: Data analysis revealed considerable heterogeneity among patients' experiences with mistletoe therapy. The importance of specific aspects became apparent, such as increased vitality to manage daily life, greater emotional and mental stability, warmth as a multidimensional phenomenon, feelings of safety and protection through mistletoe therapy, heightened self-awareness and improved self-care, as well as sensations of spiritual connectedness., Conclusions: Prior to this study it had not been shown that cancer patients using mistletoe therapy do have observations on different levels of experience. These results may lead to a deeper understanding of patients receiving mistletoe therapy, enabling them to be supported in a more holistic way both during mistletoe treatment and on their life path. Further investigations into the effects of mistletoe therapy on the emotional, mental, and spiritual level are warranted.

Objectives: Homeopathy (HOM) is a therapeutic method, which is widely used by patients and medical professionals. The medical conditions as well as the homeopathic medical products investigated vary strongly. There is an extensive amount of research, and this necessitates a bibliography that comprehensively presents the entire body of clinical evidence grouped according to medical conditions. Design: Thirty-seven online sources as well as print libraries were searched for HOM and related terms in eight languages (1980 to March 2021). We included studies that compared a homeopathic medicine or intervention with a control regarding the therapeutic or preventive outcome of a disease (classified according to International Classification of Diseases-10). The data were extracted independently by two reviewers and analyzed descriptively. Results: A total of 636 investigations met the inclusion criteria, of which 541 had a therapeutic and 95 a preventive purpose. Seventy-three percent were randomized controlled trials ( n  = 463), whereas the rest were non-randomized studies ( n  = 173). The leading comparator was placebo ( n  = 400). The type of homeopathic intervention was classified as multi-constituent or complex ( n  = 272), classical or individualized ( n  = 176), routine or clinical ( n  = 161) and isopathic ( n  = 19), or various ( n  = 8). The potencies ranged from 1X (dilution of -10,000) to 10 M (100 - 10.000 ). The included studies explored the effect of HOM in 223 medical indications. We present the evidence in an online database. Conclusions: This bibliography maps the status quo of clinical research in HOM. The data will serve for future targeted reviews, which may focus on the most studied conditions and/or homeopathic medicines, clinical impact, and the risk of bias of the included studies.

Viscum album L., popularly known as mistletoe, is well known for its anti-cancer properties, and the pharmaceutical application of hydroalcoholic dry extracts is still limited due to its low solubility in aqueous media, and physicochemical instability. The Pluronic® F127 is an amphiphilic polymer, which permits the solubilization of lipophilic and hydrophilic compounds. In this investigation, physicochemical features of hydrogel containing V. album dry extract (VADE-loaded-hydrogel) were performed by: dynamic light scattering (DLS), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and transmission electron microscopy (TEM). VADE-loaded-hydrogel presented nanometer-size micelles with volume distribution ranging from 10.58 nm to 246.7 nm, and a polydispersity index of 0.441. The sample thermal analyses (TG and DSC) showed similar decomposition curves; however, the thermal events indicated an increase in thermal stability in relation to the presence of the extract. In addition to these interesting pharmaceutical features, IC50 values of 333.40 µg/mL and >1000 µg/mL were obtained when tumor (SCC-25) and non-tumor (L929) cells were incubated with VADE-loaded-hydrogel, respectively. The optical and ultrastructural cellular analysis confirmed the tumor selectivity since the following alterations were detected only in SCC-25 cells: disorganization of plasmatic membrane; an increase of cytoplasmatic vacuole size; alteration in the cristae mitochondrial shape; and generation of amorphous cellular material. These results emphasize the promising antitumoral potential of VADE-loaded-hydrogel as an herbal drug delivery system via in vitro assays.

Viscum album is a semi-parasitic plant used for over one hundred years in complementary cancer therapy. The main commercial drugs used in cancer patients' treatment are derived from the aqueous V. album extracts, whose cytotoxic potential is mostly attributed to the aqueous soluble antitumoral metabolites. On the counterpart, ethanol solvents must be used to obtain V. album mother tinctures. This methodology permits better solubilization of phenolic compounds, among others, which present antitumoral bioactivity. Recently, the metabolomics approach revealed the influence of the host tree on the V. album subspecies differentiation. To increase the scientific information about the chemical differences related to the host trees and to clarify the seasonal influences, in this study, the metabolome of 50  V. album mother tinctures from three subspecies ( abietis , album , austriacum ) and five host trees ( Malus domestica , Quercus sp. , Ulmus carpinifolia , Pinus sylvestris , Abies alba ) was evaluated using summer and winter plant harvests. The in vitro cytotoxic activities were investigated in breast cancer cells (MDA-MB-231) and immortalized normal human keratinocytes (HaCaT). The summer V. album mother tinctures presented higher cytotoxic activity than winter ones. Among the summer samples, those prepared with V. album subsp. album were more cytotoxic than V. album subsp. abietis and subsp. V. album subsp. austriacum . The V. album harvested from Quercus petraea and Abies alba inhibited the key-glycolytic enzymes: hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK). This activity was related to a reduction in glucose uptake and lactate production, which were host-tree-time-dose-dependent. The untargeted metabolomic approach was able to discriminate the mother tinctures according to respective botanical classes and harvest season. A total of 188 metabolites were annotated under positive and negative modes. Fourteen compounds were responsible for the samples differentiation, and, to the best of our knowledge, eight were described in the Viscum album species for the first time. Our study shows the interruption of the Warburg effect as a novel antitumoral mechanism triggered by V. album mother tinctures, which is related to their metabolite profile. These results bring scientific evidence that encourages the use of V. album mother tinctures as a natural product for cancer therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Melo, Ochioni, Zancan, Oliveira, Grazi, Garrett, Holandino and Baumgartner.)

This paper introduces a deep learning based methodology for analyzing the self-assembled, fractal-like structures formed in evaporated droplets. To this end, an extensive image database of such structures of the plant extract Viscum album Quercus [Formula: see text] was used, prepared by three different mixing procedures (turbulent, laminar, and diffusion based). The proposed pattern analysis approach is based on two stages: (1) automatic selection of patches that exhibit rich texture along the database; and (2) clustering of patches in accordance with prevalent texture by means of a Dense Convolutional Neural Network. The fractality of the patterns in each cluster is verified through Local Connected Fractal Dimension histograms. Experiments with Gray-Level Co-Occurrence matrices are performed to determine the benefit of the proposed approach in comparison with well established image analysis techniques. For the investigated plant extract, significant differences were found between the production modalities; whereas the patterns obtained by laminar flow showed the highest fractal structure, the patterns obtained by the application of turbulent mixture exhibited the lowest fractality. Our approach is the first to analyze, at the pure image level, the clustering properties of regions of interest within a database of evaporated droplets. This allows a greater description and differentiation of the patterns formed through different mixing procedures., (© 2022. The Author(s).)

Purpose: Cancer-related fatigue remains one of the most prevalent and distressing symptoms experienced by cancer patients. Effective treatments for cancer-related fatigue are needed. The objective of this meta-analysis is to determine the impact of mistletoe extracts as a pharmacological treatment for the management of cancer-related fatigue., Methods: We included randomized clinical trials (RCTs) and non-randomized studies of interventions (NRSIs) in cancer patients. Inclusion criteria were cancer-related fatigue severity or prevalence as an outcome and testing of mistletoe extracts compared to control groups. We searched Medline (EuropePMC), Embase, the Cochrane Central Register of Controlled Trials, Clinicaltrials.gov, and opengrey.org through October 2020. We assessed the risk of bias using the Cochrane risk of bias tools for RCTs and NRSIs and conducted a meta-analysis., Results: We performed one meta-analysis with 12 RCTs, including 1494 participants, and one meta-analysis with seven retrospective NRSIs, including 2668 participants. Heterogeneity between the studies was high in both meta-analyses. Most studies had a high risk of bias. A random-effects model showed for RCTs a standardized mean difference of -0.48 (95% confidence interval -0.82 to -0.14; p = 0.006) and for NRSIs an odds ratio of 0.36 (95% confidence interval 0.20 to 0.66; p = 0.0008)., Conclusion: Treatment with mistletoe extracts shows a moderate effect on cancer-related fatigue of similar size to physical activity. These results need to be confirmed by more placebo-controlled trials. Future trials should investigate different treatment durations and their effect on cancer-related fatigue in post-treatment cancer survivors., Trial Registration: This meta-analysis has been registered under the PROSPERO registration number CRD42020191967 on October 7, 2020., (© 2022. The Author(s).)

Malignant ulcerating wounds or neoplastic lesions are a considerable burden for patients suffering from advanced cancer. These wounds have no effective treatment and are very difficult to manage. The present review summarizes evidence in support of a hypothesis put forward in anthroposophic medicine, which suggests a beneficial role of resin from the species Larix decidua Mill. [Pinaceae] for treating such wounds. A systematic search strategy was performed using the databases PubMed, EMBASE and SciFinder. The included publications described the chemical composition of this species, as well as in vitro , in vivo , and ex vivo experiments using plant extracts and isolated compounds. The results show that among the phytochemical classes, terpenoids were the major components of this species, especially in the resin. The summarized biological experiments revealed antimicrobial, antioxidant and anti-inflammatory effects, with promising potential for the extracts and isolated compounds. However, the molecular mechanisms and toxicological effects are as of yet not conclusively evaluated. From the data of our study, we can conclude that L. decidua might indeed have a promising potential for the treatment of malignant wounds, but definitive information that can prove its effectiveness is still lacking. We therefore suggest that future efforts should be dedicated to the evaluation of L. decidua resin's therapeutic use considering its antiseptic action and proposed wound healing properties., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Batista, Uecker, Holandino, Boylan, Maier, Huwyler and Baumgartner.)

European Pharmacopoeia monograph 2371 describes the production of homeopathic preparations. A specific efficacy of these preparations in high dilution levels is questionable in view of basic scientific principles. There is empirical evidence for such effects, for example in a Lemna-intoxication bioassay published 2010. To test the replicability and robustness of this bioassay, we conducted two experimental series (five independent blinded and randomised experiments each). The specimen of Lemna gibba L., clone-number 9352, were stressed in arsenic solution for 48 h (158 mg/L AsNa2HO4 (250 mg/L in series 2)), then grew in either As2O3 preparations produced according to Eu. Pharm. Monogr. 2371 or control solution. Comparing the area-related relative growth rate of day 3−9 (rgr 3−9) between treatment and control groups for each series showed differences that were not significant in series 1 (p = 0.10), significant in series 2 (p = 0.04) and significant in the pooled data of both series (p < 0.01). The effect direction (rgr 3−9 increase) was comparable to experiments of 2010, but the effect size was smaller, likely due to a changed light cycle. These results are not compatible with the hypothesis that the application of European Pharmacopoeia monograph 2371 results in pharmaceutical preparations without specific effects. Further studies are needed to investigate a potential mode of action explaining these effects.

Viscum album L. ( Santalaceae ), also known as European mistletoe, is a semi-parasitic plant that grows on different host trees. Our group recently demonstrated the antitumoral activity of ethanolic V. album extracts in vitro, depending on the dose and the host tree, V. album ssp abietis from Abies alba being the most active extract. The goal of this work focused on the development of a new topical formulation containing V. album extracts, evaluation of in vitro toxicity and ex vivo skin permeation assays. The Poloxamer 407 hydrogel containing 5% of dry (VA&#95;DEH) or aqueous (VA&#95;AEH) extract presented dermal compatible pH and microbiological stability for 180 days. The hydrogels flow curve presented a non-linear relation, characteristic of non-Newtonian fluids, and the mean viscosity for the VA&#95;DEH and VA&#95;AEH was 372.5 ± 7.78 and 331.0 ± 2.83 Pa.s, respectively, being statistically different (Welch's t test; p < 0.01). Additionally, WST-1 in vitro assays revealed a dose-dependent toxicity for both formulations and VA&#95;DEH presented a higher activity than the VA&#95;AEH. The promising cytotoxic potential of VA&#95;DEH lead to the ex vivo skin permeation assay with 2.73 ± 0.19 µg/cm 2 of chlorogenic acid, which permeated at 8 h, showing a transdermal potential. These in vitro results support the idea that VA&#95;DEH is a novel promising candidate for mistletoe therapy. Therefore, further in vivo and pre-clinical experiments should be performed to evaluate the safety and efficacy of this new dermic delivery system.

There are numerous diagnostic tests based on pattern formation in desiccating body fluids, where the pattern or some of its characteristics constitute the diagnostic test outcome. However, partially due to the development in different time periods, and partially due to publications in languages different from English, most of these diagnostic tests exist as separate approaches and have never been grouped, systematized, nor compared with each other. In the present mapping review, we performed a wide literature search with the aim to collect all diagnostic tests based on pattern formation in desiccating body fluids. Furthermore, we grouped the identified diagnostic tests according to their experimental protocols, type of body fluids investigated, and target conditions, and propose so for the first time a classification of different diagnostic tests based on pattern formation in desiccating body fluids. The literature search revealed 1603 publications, out of which 141 were included into the review. Following three main classification criteria (way of deposition of the fluid for desiccation, addition of reagents, and spatial restrictions during evaporation), we identified six different methods; following a further classification concerning the analyzed body fluid we identified 30 different diagnostic tests based on pattern formation in evaporating body fluids. Amongst these tests are well-known procedures such as ferning tests (tear ferning for the assessment of tear film quality, saliva and cervical mucus ferning for the detection of the fertile period, and amniotic fluid ferning for the diagnosis of fetal membrane rupture), whereas other tests are less well-established. In the latter group, the most frequently investigated body fluids were serum, saliva, and blood; the most frequently addressed target conditions were cancer, inflammation, and benign tumors. We recommend conducting further systematic reviews and meta-analyses concerning groups of methods addressing the same target condition., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Background:   Okoubaka aubrevillei is used in traditional West African medicine and in homeopathy for treatment and prevention of several gastrointestinal problems. The aim of this in vitro study was to evaluate the effect of repeated doses of two Okoubaka products (10 % ethanolic tincture, mother tincture (MT); 3 rd decimal potency, 3X) on the microbial activity of physiological human colon microbiota using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME ® ) and to investigate any preventive effect against infections with diarrhea-causing pathogens., Methods:  Upon inoculation with fecal microbiota from a healthy donor, 4 parallel proximal colon compartments of the SHIME were treated either with Okoubaka MT, Okoubaka 3X, ethanol control or blank control for 7 days. Using the Okoubaka -adapted microbial community from SHIME, 48 h challenge tests were performed with enterotoxigenic Escherichia coli (ETEC) and Salmonella enteritidis in 4 different doses (10 3 -10 8 colony forming units as typical in vivo infectious doses). Pathogen concentrations, short-chain fatty acids (SCFAs) and branched SCFA production were measured in triplicate at 0, 24 and 48 h., Results:  In the challenge tests, both Okoubaka products were able to restrict the colonization of ETEC and Salmonella at 3 of the 4 pathogen doses (except the highest doses), with a stronger anti-pathogenic effect for MT, which included a reduction of 2.0 log-units of ETEC (p < 0.0001) and 1.1 log-units of Salmonella (p < 0.0001). Total SCFA levels remained unaffected, but butyrate increased during the first 24 h (p < 0.0001 for ETEC), accompanied by decreased acetate production., Conclusion:  We observed in vitro a systemic activating effect of Okoubaka on intestinal microbiome resistance, which resulted in an anti-pathogenic effect, especially against ETEC. We hypothesize that the mode of action in vivo is also based on systemic regulative effects., Competing Interests: The research presented in this publication was financed by DHU-Arzneimittel GmbH & Co KG, Karlsruhe, Germany (DHU).Susann Buchheim-Schmidt and Petra Klement are employees of DHU.Uwe Peters received an advisory fee from DHU.Cindy Duysburgh, Pieter Van den Abbeele and Massimo Marzorati are employees of Prodigest. ProDigest received payment from DHU for conducting the research and analysing the data.Thomas Keller is owner of ACOMED statistik. ACOMED statistik received payment from DHU for statistical analysis of the study.David Martin and Stephan Baumgartner report no conflict of interest., (Thieme. All rights reserved.)

Background:  A bioassay with severely mercury-stressed duckweed ( Lemna gibba L.) had revealed growth-inhibiting effects of homeopathically potentised mercury(II) chloride ( Mercurius corrosivus, Merc-c. ). We hypothesised that effects of potentised preparations are dependent on the stress level of the organisms used in the bioassay. The aim of the present investigation was to examine the response of duckweed to potentised Merc-c. at a lower stress level., Methods:  Duckweed was moderately stressed with 2.5 mg/L mercury(II) chloride for 48 hours. Afterwards plants grew in either Merc-c. (seven different potency levels, 24x-30x) or water controls (unsuccussed or succussed water) for 7 days. Growth rates of the frond (leaf) area were determined using a computerised image-analysis system for day 0-3 and 3-7. Three independent experiments with potentised Merc-c. and three systematic negative control experiments were performed. All experiments were randomised and blinded., Results:  Unsuccussed and succussed water did not significantly differ in their effects on duckweed growth rate. The systematic negative control experiments did not yield any significant effects, thus providing evidence for the stability of the experimental system. Data from the two control groups and the seven treatment groups ( Merc-c. 24x-30x) were each pooled to increase statistical power. Duckweed growth rates for day 3-7 were enhanced ( p  < 0.05) after application of Merc-c. compared with the controls. Growth rates for day 0-3 were not influenced by the homeopathic preparations., Conclusions:  Moderately mercury-stressed Lemna gibba L. yielded evidence of growth-enhancing specific effects of Merc-c. 24x-30x in the second observation period (day 3-7). This observation is complementary to previous experiments with severely mercury-stressed duckweed, in which a decrease in growth was observed in the first observation period (day 0-3). We hypothesise that the differing results are associated with the level of stress intensity (moderate vs. severe)., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Objectives: In parts I and II of our review of physicochemical research performed on homeopathic preparations, we identified relevant publications and analyzed the data in terms of individual experiments, looking for the most promising techniques that were used in the past. In this third part, we analyze the results of the experiments seeking to extract information about the possible modes of action underpinning homeopathic preparations. Methods: We summarized the results from the 11 experimental areas previously introduced, extracting the general findings and trends. We also summarized the results in terms of specific research topics: aging, medium used for potentization, sample volume, temperature, material of potentization vessel, and, finally, the use of molecules to probe homeopathic samples. Results: We identified a number of effects that appear consistently throughout the data: Differences to controls seem to increase with: time, moderate temperature, small samples volume, and in ionic medium, whereas high temperatures seem to abolish differences to controls. Based on the present analysis, there is no consistent evidence to date for the nanoparticle hypothesis to explain specific homeopathic treatment effects. However, the quantum coherence domain hypothesis, the dynamic water cluster hypothesis, and the weak quantum theory are still contenders and need to be further assessed experimentally. Conclusions: The field requires further targeted experimentation to validate past findings reporting differences between homeopathic dilutions and controls, and to expand these findings by specifically testing the three main working hypotheses that are currently at hand.

Fermented aqueous extracts of Viscum album L. are widely used for cancer treatment in complementary medicine. The high molecular weight compounds viscotoxins and lectins are considered to be the main active substances in the extracts. However, a vast number of small molecules (≤1500 Da) is also expected to be present, and few studies have investigated their identities. In this study, a comprehensive metabolome analysis of samples of fermented aqueous extracts of V. album from two host tree species ( Malus domestica and Pinus sylvestris ), both prepared by two pharmaceutical manufacturing processes, was performed by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). A total of 212 metabolites were putatively annotated, including primary metabolites (e.g., amino acids, organic acids, etc.) and secondary metabolites (mostly phenolic compounds). A clear separation between V. album samples according to the host tree species, but not due to manufacturing processes, was observed by principal component analysis. The biomarkers responsible for this discrimination were assessed by partial least squares-discriminant analysis. Because V. album extracts from different host trees have different clinical applications, the present work highlights the possibility of characterizing the metabolome for identification and traceability of V. album fermented aqueous extracts.

Background: Viscum album L. (Santalaceae), commonly known as mistletoe, is a hemiparasitic plant traditionally used in complementary cancer treatment. Its antitumor potential is mostly attributed to the presence of aqueous soluble metabolites; however, the use of ethanol as solvent also permits the extraction of pharmacological compounds with antitumor potential. The clinical efficacy of mistletoe therapy inspired the present work, which focuses on ethanolic extracts (V. album "mother tinctures", MT) prepared from different host trees., Methods: Samples from three European subspecies (album, austriacum, and abietis) were harvested, and five different V. album-MT strains were prepared. The following phytochemical analyses were performed: thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). The proliferation assay was performed with WST-1 after incubation of tumor (Yoshida and Molt-4) and fibroblast cell lines (NIH/3 T3) with different MT concentrations (0.5 to 0.05% v/v). The cell death mechanism was investigated by flow cytometry (FACS) using Annexin V-7AAD., Results: Chemical analyses of MT showed the presence of phenolic acids, flavonoids and lignans. The MT flavonoid and viscotoxin contents (mg/g fresh weight) were highest in Quercus robur (9.67 ± 0.85 mg/g) and Malus domestica (3.95 ± 0.58 mg/mg), respectively. The viscotoxin isoform proportions (% total) were also different among the VA subspecies with a higher content of A3 in V. album growing on Abies alba (60.57 ± 2.13). The phytochemical compounds as well as the viscotoxin contents are probably related to the antitumor effects of MT. The cell death mechanisms evaluated by colorimetric and FACS methodologies involved necrotic damage, which was host tree-, time- and dose- dependent, with different selectivity to tumor cells. Mother tincture from V. album ssp. abietis was the most effective at inducing in vitro cellular effects, even when incubated at the smallest concentration tested, probably because of the higher content of VT A3., Conclusion: Our results indicate the promising antitumor potential of Viscum album ethanolic extracts and the importance of botanical and phytochemical characterization for in vitro anti-proliferative effects.

The aim of the present study was to investigate if patterns obtained from evaporating droplets of pharmaceutical preparations reveal the impact of succussion on such medicinal products. For this purpose, five pharmaceutical preparations (Echinacea 10 -2 , Baptisia 10 -3 , Baptisia 10 -4 , Luffa 10 -4 , and Spongia 10 -6 ) were prepared according to the European Pharmacopoeia guidelines for the production of homeopathic remedies, in three variants each: with varying numbers of succussion strokes (i) 100, (ii) 10 (succussed samples), and (iii) zero (gently mixed, unsuccussed sample). System stability was studied by means of systematic positive control experiments. Patterns were evaluated by means of computerized image analysis regarding grey level distribution, texture, and fractality. For all investigated pharmaceutical preparations, significant differences were found between the succussed and gently mixed samples; whereas, all three samples (prepared with 100, 10 and zero succussion strokes) could be significantly differentiated for Luffa 10 -4 and Spongia 10 -6 for one image evaluation parameter each. Control experiments showed a reasonable stability of the experimental set-up.

Objectives: In Part 1 of the review of physicochemical research performed on homeopathic preparations the authors identified relevant publications of sufficient reporting quality for further in-depth analysis. In this article, the authors analyze these publications to identify any empirical evidence for specific physicochemical properties of homeopathic preparations and to identify most promising experimental techniques for future studies. Methods: After an update of the literature search up to 2018, the authors analyzed all publications in terms of individual experiments. They extracted information regarding methodological criteria such as blinding, randomization, statistics, controls, sample preparation, and replications, as well as regarding experimental design and measurement methods applied. Scores were developed to identify experimental techniques with most reliable outcomes. Results: The publications analyzed described 203 experiments. Less than 25% used blinding and/or randomization, and about one third used adequate controls to identify specific effects of homeopathic preparations. The most promising techniques used so far are nuclear magnetic resonance (NMR) relaxation, optical spectroscopy, and electrical impedance measurements. In these three areas, several sets of replicated high-quality experiments provide evidence for specific physicochemical properties of homeopathic preparations. Conclusions: The authors uncovered a number of promising experimental techniques that warrant replication to assess the reported physicochemical properties of homeopathic preparations compared with controls. They further discuss a range of experimental aspects that highlight the many factors that need to be taken into consideration when performing basic research into homeopathic potentization. For future experiments, the authors generally recommend using succussed (vigorously shaken) controls, or comparing different homeopathic preparations with each other to reliably identify any specific physicochemical properties.

Background: We developed a bioassay with mercury-stressed duckweed ( Lemna gibba L.) to study potential effects of homeopathically potentised mercury(II) chloride ( Mercurius corrosivus [ Merc-c. ]). The response of this bioassay to homeopathic treatments as a function of stress intensity was also of interest., Methods: Duckweed was severely stressed with mercury(II) chloride for 48 hours. Afterwards plants grew in either Merc-c. (seven different potency levels, 24x to 30x) or water controls (unsuccussed and succussed water) for 7 days. Growth rates of the frond (leaf) area were determined using a computerised image analysis system for different time intervals between the measurements on days 0, 3 and 7. Three independent experiments with potentised Merc-c. each were evaluated. Additionally, three water control experiments were analysed to investigate the stability of the experimental set-up (systematic negative control [SNC] experiments). All experiments were randomised and blinded., Results: Unsuccussed and succussed water did not significantly differ in terms of duckweed growth rate. The SNC experiments did not yield any significant effects, providing evidence for the stability of the experimental system. Data from the two control groups and the seven treatment groups ( Merc-c. 24x-30x) were each pooled to increase the statistical power. Duckweed growth rates for day 0 to 3 were reduced ( p  < 0.05) after application of Merc-c. compared with the controls. Growth rates for day 3 to 7 were not influenced by the homeopathic preparations., Conclusions: The present test system with Lemna gibba L. that was severely stressed by mercury yielded evidence for specific effects of Merc-c. 24x to 30x, namely a growth reduction in the first time period (day 0-3). This is in contrast to former experiments with slightly arsenic-stressed duckweed, where a growth increase was observed in the second time period (day 2-6). We hypothesise that the differing results are associated with the level of stress intensity (severe versus slight)., Competing Interests: None declared., (The Faculty of Homeopathy.)

Background: Evaporation-induced pattern formation in droplets has been applied to test effects of high potencies. Here we propose for the first time the droplet evaporation method (DEM) as a tool to characterize low potencies on a qualitative and quantitative basis., Materials and Methods: The present investigation consisted of: (1) screening of 18 different substances of mineral, vegetal, and animal origin in the 1x to 6x potency range; choice of four substances with characteristic pattern-forming properties; (2) replication experiments aiming at the differentiation of four homeopathic preparations at the same potency levels (2x-6x); and (3) control experiments performed on three preparations. The DEM experimental protocol consisted of the evaporation of droplets of the analyzed potency per se , placed on microscope slides and in controlled conditions. The resulting patterns were photographed and subjected to computerized image analysis., Results: The screening experiments yielded a wide variety of patterns. Homeopathic preparations of mineral origin showed the largest variety of forms, whereas potencies of vegetal origin mostly created dendritic patterns, probably due to diffusion-limited aggregation. The here-analyzed image analysis variables (gray-level distribution, entropy, and inverse difference moment) allowed a highly significant differentiation of patterns prepared from four substances ( Echinacea , Baptisia , Luffa , and Spongia ) at the same potency levels in the range 2x to 4x, whereas patterns obtained from potencies 5x and 6x could no longer be differentiated and resembled the pattern of pure solute (purified water). The control experiments showed reasonable experimental model stability., Conclusions: DEM seems to be a promising tool for qualitative phenomenological characterization of homeopathic preparations in low potency. We propose the application of the current experimental model for investigating further research topics in this field, such as the comparison of potencies versus simple dilutions or the contribution of component remedies to the patterns formed by homeopathic combination medicines., Competing Interests: None declared., (The Faculty of Homeopathy.)

Objectives: From a pharmaceutical point of view, we see a need to develop stable preclinical test systems to identify and investigate effects of potentized remedies as used in Anthroposophic Medicine and Homeopathy. We evaluated a plant bioassay regarding its capacity to distinguish homeopathic remedies from placebo, applied as sucrose pillules., Methods: Pea seed (Pisum sativum L) was soaked for 24 hours in water with dissolved homeopathic or placebo pillules, or in water only. Shoot length was measured 14 days after planting and treatment groups were compared by analysis of variance (ANOVA). The stability of the system was validated by systematic negative control experiments., Results: The system is suitable to test a common application form - sucrose pillules - of a potentized preparation without influence of the pharmaceutical carrier substance. A screening of 13 potentized preparations revealed Calcium carbonicum to affect pea shoot growth (p < 0.05). Three independent series of main experiments were performed with potentized Calcium carbonicum to assess reproducibility. Meta-analysis of all data revealed significant effects of Calcium carbonicum 12c and 30c on pea shoot growth (p < 0.05), which were however dependent on the date of experiment and/or the experimental series., Conclusions: Potentized Calcium carbonicum, applied as sucrose pillules, influenced pea shoot growth in the assay investigated. However, due to the small effect size and due to the modulation of the effects by still unknown external factors, further optimization of this bioassay is necessary to be used in pharmaceutical quality control or in investigating the biological or pharmaceutical mode of action of potentized preparations., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Methods based on phase-transition-induced pattern formation (PTPF) are increasingly used in medical research. Frequent application fields are medical diagnosis and basic research in homeopathy. Here, we present a systematic review of experimental studies concerning PTPF-based methods applied to homeopathy research. We also aimed at categorizing the PTPF methods included in this review., Materials and Methods: Experimental studies were collected from scientific databases (PubMed, Web of Science, Russian eLibrary) and from experts in the research field in question, following the PRISMA guidelines. The studies were rated according to pre-defined scientific criteria., Results: The review included 15 experimental studies. We identified seven different PTPF methods applied in 12 experimental models. Among these methods, phase-transition was triggered through evaporation, freezing, or solution, and in most cases led to the formation of crystals. First experimental studies concerning the application of PTPF methods in homeopathic research were performed in the first half of the 20th century; however, they were not continued in the following years. Only in the last decade, different research groups re-launched the idea, introducing new experimental approaches and computerized pattern evaluation techniques. The here-identified PTPF methods are for the first time proposed to be classified as one group of methods based on the same basic physical phenomenon., Conclusions: Although the number of experimental studies in the area is still rather limited, the long tradition in the application of PTPF methods and the dynamics of the present developments point out the high potential of these methods and indicate that they might meet the demand for scientific methods to study potentized preparations., Competing Interests: None., (The Faculty of Homeopathy.)

Background: Plant-based test systems have been described as a useful tool for investigating possible effects of homeopathic preparations. The last reviews of this research field were published in 2009/2011. Due to recent developments in the field, an update is warranted. Publications on plant-based test systems were analysed with regard to publication quality, reproducibility and potential for further research., Methods: A literature search was conducted in online databases and specific journals, including publications from 2008 to 2017 dealing with plant-based test systems in homeopathic basic research. To be included, they had to contain statistical analysis and fulfil quality criteria according to a pre-defined manuscript information score (MIS). Publications scoring at least 5 points (maximum 10 points) were assumed to be adequate. They were analysed for the use of adequate controls, outcome and reproducibility., Results: Seventy-four publications on plant-based test systems were found. Thirty-nine publications were either abstracts or proceedings of conferences and were excluded. From the remaining 35 publications, 26 reached a score of 5 or higher in the MIS. Adequate controls were used in 13 of these publications. All of them described specific effects of homeopathic preparations. The publication quality still varied: a substantial number of publications (23%) did not adequately document the methods used. Four reported on replication trials. One replication trial found effects of homeopathic preparations comparable to the original study. Three replication trials failed to confirm the original study but identified possible external influencing factors. Five publications described novel plant-based test systems. Eight trials used systematic negative control experiments to document test system stability., Conclusions: Regarding research design, future trials should implement adequate controls to identify specific effects of homeopathic preparations and include systematic negative control experiments. Further external and internal replication trials, and control of influencing factors, are needed to verify results. Standardised test systems should be developed., Competing Interests: None., (The Faculty of Homeopathy.)

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. The quality of publications was seen to increase sharply from 2000 onward, whereas before 2000, only 12 (13%) publications were rated as "high quality" (MIS ≥7.5); 44 (48%) publications were rated as "high quality" from 2000 onward. Countries with most publications were Germany (n = 42, 23%), France (n = 29, 16%), India (n = 27, 15%), and Italy (n = 26, 14%). Techniques most frequently used were electrical impedance (26%), analytical methods (20%), spectroscopy (20%), and nuclear magnetic resonance (19%)., Conclusions: Physicochemical research into homeopathic preparations is increasing both in terms of quantity and quality of the publications.

Background: A previous study reported a significant statistical interaction between experiment date and treatment effect of Argentum nitricum 14x-30x on the growth rate of duckweed (Lemna gibba L.). The aim of the present study was to investigate the stability of the test system and intra-laboratory reproducibility of the effects found., Methods: Duckweed was treated with A. nitricum potencies (14x-30x) as well as succussed and unsuccussed water controls. The outcome parameter area-related growth rate for day 0-7 was determined by a computerised image analysis system in two series of independent randomised and blinded experiments. Systematic negative control (SNC) experiments were carried out to investigate test system stability. Statistical analysis was performed with full two-way analysis of variance (ANOVA) and protected Fisher's Least Significant Difference (LSD) test., Results: In the first repetition series we found a significant treatment effect (p = 0.016), while in the second series no effect was observed. The negative control experiments showed that the experimental system was stable. An a posteriori subgroup analysis concerning gibbosity revealed the importance of this growth state of L. gibba for successful reproduction of the statistically significant interaction in the original study; flat: no interaction (p = 0.762); slight gibbosity: no interaction (p = 0.356); medium gibbosity: significant interaction (p = 0.031), high gibbosity: highly significant interaction (p = 0.005)., Conclusions: With the original study design (disregarding gibbosity status of L. gibba) results of the original study could not be reproduced sensu stricto. We conclude that the growth state gibbosity is crucial for successful reproduction of the original study. Different physiological states of the test organisms used for bioassays for homeopathic basic research must carefully be considered., (Copyright © 2017 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.)

This paper presents a discovery: the change of the outline shape of mistletoe ( Viscum album ssp. album ) berries in vivo and in situ during ripening. It was found that a plant organ that is usually considered to merely increase in size actually changes shape in a specific rhythmic fashion. We introduce a new approach to chronobiological research on a macro-phenotypic scale to trace changes over long periods of time (with a resolution from hours to months) by using a dynamic form-determining parameter called Lambda (λ). λ is known in projective geometry as a measure for pertinent features of the outline shapes of egg-like forms, so called path curves. Ascertained circadian changes of form were analyzed for their correlation with environmental factors such as light, temperature, and other weather influences. Certain weather conditions such as sky cover, i.e., sunshine minutes per hour, have an impact on the amplitude of the daily change in form. The present paper suggests a possible supplement to established methods in chronobiology, as in this case the dynamic of form-change becomes a measurable feature, displaying a convincing accordance between mathematical rule and plant shape.

Background: Polycrystalline structures formed inside evaporating droplets of different biological fluids have been shown sensitive towards various influences, including ultra high dilutions (UHDs), representing so a new approach potentially useful for basic research in homeopathy. In the present study we tested on a wheat seed model Zincum metallicum 30c efficacy versus lactose 30c and water., Materials and Methods: Stressed and non-stressed wheat seeds were watered with the three treatments. Seed-leakage droplets were evaporated and the polycrystalline structures formed inside the droplet residues were analyzed for their local connected fractal dimensions (LCFDs) (measure of complexity) using the software ImageJ., Results: We have found significant differences in LCFD values of polycrystalline structures obtained from stressed seeds following the treatments (p<0.0001); Zincum metallicum 30c lowered the structures' complexity compared to lactose 30c and water. In non-stressed seeds no significant differences were found., Conclusions: The droplet evaporation method (DEM) might represent a potentially useful tool in basic research in homeopathy. Furthermore our results suggest a sensitization of the stressed model towards the treatment action, which is conforming to previous findings., (Copyright © 2015 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.)

Objectives: The objective was to evaluate homeopathic basic research studies that use plant-based bioassays. With this in view, a compilation was made of the findings of three systematic literature reviews covering plant-based bioassays in the three fields of healthy, abiotically, or biotically stressed plants. This compilation focused on investigations using advanced experimental methods and detailed descriptions, also with the aim of supporting the design of future experiments., Methods: Publications included had to report on studies into the effects of homeopathic preparations on whole plants, seeds, plant parts and cells. Outcomes had to be measured by established procedures and statistically evaluated. A Manuscript Information Score (MIS) was applied using predefined criteria to identify publications with sufficient information for adequate interpretation (MIS ≥ 5). Additional evaluation focused on the use of adequate controls to investigate specific effects of homeopathic preparations, and on the use of systematic negative control (SNC) experiments to ensure the stability of the bioassay. Only a fraction of the studies reported here were performed with 'ultra high' dilutions, whereas other studies were performed with moderate or high dilutions., Results: A total of 157 publications were identified, describing a total of 167 experimental studies. 84 studies included statistics and 48 had a MIS ≥ 5, thus allowing adequate interpretation. 29 studies had adequate controls to identify specific effects of homeopathic preparations, and reported significant effects of decimal and centesimal homeopathic potencies, including dilution levels beyond Avogadro's number. 10 studies reported use of SNC experiments, yielding evidence for the stability of the experimental set-up., Conclusion: Plant models appear to be a useful approach for investigating basic research questions relating to homeopathic preparations, but more independent replication trials are needed in order to verify the results found in single experiments. Adequate controls and SNC experiments should be implemented on a routine basis to exclude false-positive results., (Copyright © 2015 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.)

Background: Macrophages are highly versatile cells that play an important role in tumour microenvironment. Tumour associated macrophages (TAMs) have been linked to both, good or bad prognosis of several cancer types depending on their number, composition and polarization. Viscum album lipophilic extract (VALE) contains several pentacyclic triterpenes known to modulate the activity of monocytes and other immune cells and to exhibit anticancer properties. In our in vitro study, we investigated the effect of tumour cell lines on macrophage polarization and monocyte chemotactic transmigration and examined the modulatory potential of VALE and its predominant triterpene oleanolic acid (OA)., Methods: Human peripheral blood monocytes were differentiated into monocyte derived macrophages (MDM) using M-CSF and polarized into M1 by IFN-γ and LPS and into M2 macrophages by IL-4 and IL-13 or by co-culture with two different tumour cell lines. Polarized macrophages were subsequently treated with VALE or OA. Phenotypic markers and cytokines were assessed by flow cytometry and immunoanalysis. Migration of human peripheral blood monocytes induced by monocyte chemotactic protein-1 (MCP-1) or supernatants of different tumour cell lines under the influence of VALE or OA was measured in a chemotaxis transmigration assay., Results: In vitro polarized M1 and M2 type macrophages revealed specific phenotypic patterns and tumour cell co-cultured MDM displayed ambiguous phenotypes with M1 as well as M2 associated markers. VALE and OA showed modest influence on cell surface marker profile and cytokine expression of tumour cell co-cultured macrophages. All tumour cell supernatants markedly enhanced the migratory activity of monocytes. VALE and OA significantly inhibited MCP-1 induced monocyte transmigration, whereas monocyte migration initiated by tumour cell derived supernatants was not affected., Conclusions: In our study we reconfirmed that co-culture with different tumour cell lines can result in a mixed macrophage phenotype with M1 as well as M2 patterns, a finding that is important for a better understanding of tumour microenvironment functions. Moreover, we demonstrated that VALE shows slight immunomodulatory effects on tumour cell co-cultured macrophages and modulates monocyte chemotactic transmigration in vitro, indicating promising possibilities of triterpenes from Viscum album L. to contribute in a multimodal concept of anti-cancer therapy in future. Our data contribute to an understanding of monocyte function and macrophage polarization in vitro and of the possibility to influence their behaviour by triterpene containing mistletoe extracts.

Background: Reproducibility of basic research investigations in homeopathy is challenging. This study investigated if formerly observed effects of homeopathically potentised gibberellic acid (GA3) on growth of duckweed (Lemna gibba L.) were reproducible., Methods: Duckweed was grown in potencies (14x-30x) of GA3 and one time succussed and unsuccussed water controls. Outcome parameter area-related growth rate was determined by a computerised image analysis system. Three series including five independent blinded and randomised potency experiments (PE) each were carried out. System stability was controlled by three series of five systematic negative control (SNC) experiments. Gibbosity (a specific growth state of L. gibba) was investigated as possibly essential factor for reactivity of L. gibba towards potentised GA3 in one series of potency and SNC experiments, respectively., Results: Only in the third series with gibbous L. gibba L. we observed a significant effect (p = 0.009, F-test) of the homeopathic treatment. However, growth rate increased in contrast to the former study, and most biologically active potency levels differed. Variability in PE was lower than in SNC experiments. The stability of the experimental system was verified by the SNC experiments., Conclusions: Gibbosity seems to be a necessary condition for reactivity of L. gibba to potentised GA3. Further still unknown conditions seem to govern effect direction and the pattern of active and inactive potency levels. When designing new reproducibility studies, the physiological state of the test organism must be considered. Variability might be an interesting parameter to investigate effects of homeopathic remedies in basic research., (Copyright © 2014 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.)

Background: Given the importance of complementary and alternative medicine (CAM) to cancer patients, there is an increasing need to learn more about possible interactions between CAM and anticancer drugs. Mistletoe (Viscum album L.) belongs to the medicinal herbs that are used as supportive care during chemotherapy. In the in vitro study presented here the effect of standardized mistletoe preparations on the cytostatic and cytotoxic activity of several common conventional chemotherapeutic drugs was investigated using different cancer cell lines., Methods: Human breast carcinoma cell lines HCC1937 and HCC1143 were treated with doxorubicin hydrochloride, pancreas adenocarcinoma cell line PA-TU-8902 with gemcitabine hydrochloride, prostate carcinoma cell line DU145 with docetaxel and mitoxantrone hydrochloride and lung carcinoma cell line NCI-H460 was treated with docetaxel and cisplatin. Each dose of the respective chemotherapeutic drug was combined with Viscum album extract (VAE) in clinically relevant concentrations and proliferation and apoptosis were measured., Results: VAE did not inhibit chemotherapy induced cytostasis and cytotoxicity in any of our experimental settings. At higher concentrations VAE showed an additive inhibitory effect., Conclusions: Our in vitro results suggest that no risk of safety by herb drug interactions has to be expected from the exposition of cancer cells to chemotherapeutic drugs and VAE simultaneously.

Extracts from European mistletoe (Viscum album L.) developed in anthroposophic medicine are based on specific pharmaceutical procedures to enhance remedy efficacy. One such anthroposophic pharmaceutical process was evaluated regarding effects on cancer cell toxicity in vitro and on colchicine tumor formation in Lepidium sativum. Anthroposophically processed Viscum album extract (APVAE) was produced by mixing winter and summer mistletoe extracts in the edge of a high-speed rotating disk and was compared with manually mixed Viscum album extract (VAE). The antiproliferative effect of VAE/APVAE was determined in five cell lines (NCI-H460, DU-145, HCC1143, MV3, and PA-TU-8902) by WST-1 assay in vitro; no difference was found between VAE and APVAE in any cell line tested (P > 0.14). Incidence of colchicine tumor formation was assessed by measurement of the root/shoot-ratio of seedlings of Lepidium sativum treated with colchicine as well as VAE, APVAE, or water. Colchicine tumor formation decreased after application of VAE (-5.4% compared to water, P < 0.001) and was even stronger by APVAE (-8.8% compared to water, P < 0.001). The high-speed mistletoe extract mixing process investigated thus did not influence toxicity against cancer cells but seemed to sustain morphostasis and to enhance resistance against external noxious influences leading to phenomenological malformations.

Homeopathic remedies are produced by potentising, that is, the serial logarithmic dilution and succussion of a mother tincture. Techniques like ultraviolet spectroscopy, nuclear magnetic resonance, calorimetry, or thermoluminescence have been used to investigate their physical properties. In this study, homeopathic centesimal (c) potencies (6c to 30c) of copper sulfate, Hypericum perforatum, and sulfur as well as succussed water controls were prepared. Samples of these preparations were exposed to external physical factors like heat, pressure, ultraviolet radiation, or electromagnetic fields to mimic possible everyday storage conditions. The median transmissions from 190 nm to 340 nm and 220 nm to 340 nm were determined by ultraviolet light spectroscopy on five measurement days distributed over several months. Transmissions of controls and potencies of sulfur differed significantly on two of five measurement days and after exposure to physical factors. Transmissions of potencies exposed to ultraviolet light and unexposed potencies of copper sulfate and Hypericum perforatum differed significantly. Potency levels 6c to 30c were also compared, and wavelike patterns of higher and lower transmissions were found. The Kruskal-Wallis test yielded significant differences for the potency levels of all three substances. Aiming at understanding the physical properties of homeopathic preparations, this study confirmed and expanded the findings of previous studies.

Photographs of mistletoe (Viscum album L.) berries taken by a permanently fixed camera during their development in autumn were subjected to an outline shape analysis by fitting path curves using a mathematical algorithm from projective geometry. During growth and maturation processes the shape of mistletoe berries can be described by a set of such path curves, making it possible to extract changes of shape using one parameter called Lambda. Lambda describes the outline shape of a path curve. Here we present methods and software to capture and measure these changes of form over time. The present paper describes the software used to automatize a number of tasks including contour recognition, optimization of fitting the contour via hill-climbing, derivation of the path curves, computation of Lambda and blinding the pictures for the operator. The validity of the program is demonstrated by results from three independent measurements showing circadian rhythm in mistletoe berries. The program is available as open source and will be applied in a project to analyze the chronobiology of shape in mistletoe berries and the buds of their host trees.