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Malignant pleural mesothelioma (MPM) is a rare cancer with a dismal prognosis. Viscum album extracts (VAE) have strong immune stimulatory properties, cytotoxic effects, can downregulate cancer genes and inhibit angiogenesis. VAE are often used as an adjunct treatment in cancer patients but have rarely been investigated in MPM. Helleborus niger extracts (HNE) have been used in anticancer therapy since antiquity, and also show tumor specific cytotoxic effects. We present a case of a 64-year old woman with epithelioid MPM of the right chest with node involvement (T2N1M0, stage III). Deciding against the recommended radio-chemotherapy, surgery and pleurodesis, she opted for an integrative treatment approach and was treated with VAE and HNE. After 6 weeks’ treatment, the pleural and nodal MPM manifestations were reduced by about 15%. Subsequent tumor growth was slow, and the patient remained in good health, enabling her to remain physically active until shortly before her death 56 months after the initial diagnosis. This is a rare case of an MPM patient not receiving any standard anticancer treatment; it still shows an extraordinary long survival and good performance status. We presume that VAE and HNE might had an impact on this clinically relevant outcome and therefore should be further investigated in MPM.

Background The therapeutic use of Helleborus niger L. is manifold due to its specific phytochemical composition. Two compound groups, the ranunculin derivates including protoanemonin and the steroidal saponins, are also associated with toxicity (genotoxicity, disintegration of membrane structures). Therefore, in vitro investigations were performed on safety aspects of a Helleborus niger aqueous fermented extract (HNE). In addition its therapeutic potential against various cancer cell lines was assessed to gain insight into the respective mechanisms of action. Methods To evaluate the safe use of HNE, Ames and hemolytic tests were carried out. Two angiogenesis assays in 2D and 3D design were conducted to assess the anti-angiogenetic potential, for which human umbilical vein endothelial cells (HUVEC) were chosen. A panel of tumor cell lines was used in 2D and 3D proliferation assays as well in the migration- and invasion-assay. All investigations were performed with HNE compared to reference substances. The 2D proliferation assay was additionally performed with isolated compounds of HNE (characteristic steroidal saponins). Results HNE did not exhibit any genotoxic potential. Concentrations up to 10 μl/ml were classified as non-hemolytic. HNE exerted anti-angiogenetic effects in HUVEC and anti-proliferative effects in five cancer cell lines, whereas hellebosaponin A and D as well macranthosid I did not show comparable effects neither singly nor in combination. Due to the inherent instability of protoanemonin in isolated form, parallel investigations with protoanemonin could not be performed. HNE (600–1000 μg/ml) inhibited the migration of certain cancer cells by > 80% such as Caki-2, DLD-1 and SK-N-SH. Conclusion HNE exhibit neither genotoxic nor hemolytic potential. The present investigations verify the anti-angiogenetic effects on HUVEC, the anti-proliferative effects and migration-inhibiting properties on tumor cells. The lower effect of the relevant steroidal saponins compared to the whole extract underlines the fact that the latter is more effective than a blend of isolated pharmacologically active components. Electronic supplementary material The online version of this article (10.1186/s12906-019-2517-5) contains supplementary material, which is available to authorized users.

In dieser Fallbeschreibung wird ein individuelles integratives Behandlungskonzept bei einer palliativen Patientin mit metastasiertem Mammakarzinom vorgestellt. Im Mittelpunkt steht dabei Helleborus niger (Christrose), eine in der komplementaren Onkologie bisher wenig beachtete Heilpflanze. Im vorliegenden Fall wurde Helleborus niger D12 in Form von taglichen Kaltinhalationen zunachst zur symptomatischen Behandlung von Dyspnoe und Reizhusten bei Pleuraerguss eingesetzt. Daruber hinaus verabreichten wir taglich subkutane Injektionen zur Immunmodulation wie auch zur Unterstutzung der Diurese. In Kombination mit anderen komplementaren Therapieprinzipien liesen sich zusatzlich gute Effekte hinsichtlich Kachexie, Muskelabbau, Schlaflosigkeit, Angst und Unruhe erzielen, die alle palliativmedizinisch relevant sind. Dabei waren weder Nebenwirkungen noch Wechselwirkungen mit chemischen oder pflanzlichen Stoffen zu verzeichnen. Nach 9-monatiger Therapie war klinisch wie sonografisch kein Pleuraerguss mehr nachweisbar. Auch die ubrigen Tumormanifestationen befanden sich in Regression, was mit einer deutlichen Stabilisierung der Patientin und Verbesserung ihrer Lebensqualitat einherging. Es lohnt daher, Helleborus niger in integrative Behandlungskonzepte aufzunehmen und durch breiteren klinischen Einsatz weitere Wirkmechanismen zu eruieren.

The aerial parts of the medicinal plant Helleborus niger L. comprise a substantial number of constituents with only few of them identified so far. To expand the knowledge of its secondary metabolite profile, extracts from H. niger leaves and stems were investigated by liquid chromatography/tandem mass spectrometry (LC/MS(n) ). Specific identification strategies using LC/MS are established and discussed in detail. The leaves turned out to contain acylated and non-acylated quercetin and kaempferol oligoglycosides, protoanemonin and its precursor ranunculin, β-ecdysone, and a variety of steroidal saponins, mainly in the furostanol form. The sapogenins were elucidated as of sarsasapogenyl, diosgenyl, and macranthogenyl structures, and confirmed by comparison with the respective reference compounds. The secondary metabolite profiles were almost identical in both plant parts except that the stems lacked kaempferol derivatives and some saponins. The ranunculin derivatives and β-ecdysone were found in both plant parts. Correlations between the location of the compound groups and the plant's defense strategies are proposed. Additionally, the role of the detected secondary metabolites as protective substances against exogenic stress and as a defense against herbivores is discussed.

Melampus is a seer-healer of Greek myth attributed with having healed the young princesses of Argos of madness. Analysis of this legend and its sources sheds light on the early stages of the “medicalizing” shift in the history of ancient Greek medicine. Retrospective psychological diagnosis suggests that the descriptions of the youths’ madness rose from actual observation of behavioral and mental disorders. Melampus is credited with having healed them by administering hellebore. Pharmacological analysis of botanical specimens proves that Helleborus niger features actual neurological properties effective in the treatment of mental disorders. The discussion aims at examining the rational aspects of the treatment of mental conditions in Greco-Roman antiquity.

Ethnopharmacological relevance In Romanian folk medicine, Helleborus niger L. is used for the treatment of rheumatoid arthritis or viral infections and in complementary therapy, especially in anthroposophic medicine (AM), where the plant is administered as an adjuvant to treat malignant diseases. In the present study, we investigated the differential cytotoxic effects of H. niger on human tumour and healthy cells of the human immune system in vitro . Material and methods Protoanemonin and saponins, as significant constituents of H. niger extracts, were quantified in five individual batches using validated HPLC methods. Further, the impact of H. niger on proliferation capacity (MTT assay) as well as on apoptosis and necrosis induction in a panel of tumour cell lines and human lymphocytes (combined annexin V and propidium iodide staining) was monitored. In addition, NK cell function (degranulation-CD107a assay and IFN-gamma secretion) was also investigated since these immunocompetent cells are important for the control of malignancies within the human body. Results Extracts of H. niger induced proliferation inhibition not only of lymphoblastic leukaemia cells (MOLT4; IC 50 : 171 µg/mL) but also of myosarcoma (SK-UT-1b; IC 50 : 304 µg/mL) and melanoma cells (HT-144; IC 50 : 569 µg/mL) due to the induction of apoptosis. Purified T cells or NK cells were significantly affected through the presence of high H. niger concentrations while bulk lymphocytes were not affected. NK cells’ anti-tumour functions expressed by degranulation capacity as well as IFN-y production were unaffected by the presence of the H. niger extract. Since protoanemonin and saponins have been reported in the literature to exert cytotoxic effects, their content was also determined. Conclusions H. niger extracts exhibit differential cytotoxicity towards tumour cell lines and healthy human T- and NK-cells.

Steroidal saponins comprise a substantial part of the secondary metabolite spectrum in the medicinal plant Helleborus niger L. (black hellebore). The saponin fraction from the roots was investigated by LC-MS(n) resulting in 38 saponins and β-ecdysone. Nine diosgenyl-type glycosides, mainly furostanols consisting of the aglycones diosgenin, macranthogenin, sceptrumgenin, and sarsasapogenin were accompanied by 5 diosgenyl-type saponins exhibiting an aglycone with an additional OH group. However, the most relevant compounds were 24 acetylated polyhydroxy saponins including hellebosaponins A and D. The enzymes glucuronidase, β-glucosidase, and pectinase were used to obtain an idea on potential fermentative transformation reactions by incubation of the isolated model saponins macranthosid I and hellebosaponin A. In a second step, aqueous H. niger extracts containing a much greater range of saponins were monitored during fermentation and 12months of storage. The metabolites were examined and assigned by LC-MS(n) and targeted extracted ion current (EIC) scan analyses. Good agreement was found among the results from the model compounds and the whole aqueous fermented extracts. The native diosgenyl-type furostanol saponins were converted to spirostanols under scission of hexoses. Alteration of the acetylated polyhydroxy saponins, exclusively spirostanols, took place following cleavage of acetyl groups and terminal deoxyhexoses. Most interestingly, the pentoses of the sugar chain at C(1) were not affected. Conversion of acetylated polyhydroxy saponins resulted in a final structure type which was stable and detectable, even after 12months of fermentation and storage.

Phytochemical investigation of Helleborus niger L. (Ranunculaceae) leaf methanol extract allowed to isolate a phenolic glucoside derivative and two flavonoid glycosides characterized as phenyllactic acid 2-O-β-D-glucopyranoside (1), quercetin 3-O-2-(E-caffeoyl)-α-L-arabinopyranosyl-(1 → 2)-β-D-glucopyranoside-7-O-β-D-glucopyranoside (2), and kaempferol 3-O-α-L-arabinopyranosyl-(1 → 2)-β-D-galactopyranoside-7-O-β-D-glucopyranoside (3), respectively. Compounds 1 and 2 were isolated for the first time and their structural characterization was obtained on the basis of extensive NMR spectral studies.

Helleborus niger L (HN) is widely used in the treatment of diabetes mellitus in the Indian traditional system of medicine. Therefore, the present study was done to evaluate the antihyperglycemic, antihyperlipidemic and antioxidant activities of ethanol extract of HN root (EHN) in streptozotocin (STZ) - nicotinamide (NC) induced diabetic rats. in vitro α-amylase inhibition assay, normoglycemic study and oral glucose tolerance test were carried out in different root extracts of HN. Antihyperglycemic effect was assessed in EHN using diabetic rats, which was identified as the most effective extract by initial screening. EHN (50, 100 and 200 mg/kg) and glibenclamide (5 mg/kg) orally administered daily for 28 days and the animal was observed in next 14 days. Fasting blood glucose (FBG) and body weight was determined weekly basis up to 42 days. On the 42nd day, various biochemical parameters were estimated. The three doses of EHN shows significantly decrease (p

Helleborus niger , also known as Christmas Rose, belongs to the family of Ranunculaceae, a family of flowering plants with about 2500 different species. In anthroposophic medicine H. niger is used as adjuvant drug in the treatment of brain tumors, leukemia, lymphoma and prostate cancer. Although it is widely applied in these malignancies, there are no clinical or preclinical data regarding the effects of H. niger in vivo , ex vivo or in vitro . For this purpose, we investigated the cytotoxic effects of four different standardized aqueous H. niger extracts on various cancer cell lines. We used one whole plant extract, one root extract, one leave extract and one containing only the blossom of H. niger . After 4 h no significant LDH release was measured, thus excluding an unspecific, necrotic damage to the cell membrane. After 24 h a dose-dependent inhibition of proliferation was determined and after 48 h a distinction into early and late apoptotic cells was detected via annexin/PI staining. The cell cycle analysis revealed characteristic hypodiploid DNA after 72 h, once more identifying apoptosis as a cause of the cell death. Apoptotic cell death was detected in the Burkitt-like lymphoma cell line BJAB, the leukemia cell lines NALM-6, Sup-B-15 and REH and the melanoma cell line MEL-HO. Hereby, the whole plant and the root extract revealed the strongest apoptosis induction. In western blot analysis an activation of Caspase-3, the main executioner caspase of apoptosis, could be found after 36-h incubation with the extract. A breakdown of the mitochondrial membrane potential and dose-dependent mitochondrial permeability transition were determined after 48 h, revealing that apoptosis is executed via the mitochondrial pathway. Furthermore, we could find a decreased apoptosis induction in BCL-2 overexpressing melanoma cells. The dependency on Bcl-2 expression is another sign of apoptosis via the mitochondrial pathway. Finally, we evaluated the effect on primary cells ex vivo . Interestingly, we could show a significant apoptosis induction in primary leukemia cells from patients with ALL or AML in childhood, which were partly poor responding to the anthracyclines doxorubicin and daunorubicin. In Nalm6 cells resistant to vincristine and paclitaxel, apoptosis induction by H. niger was as high in the control than in the resistant cell line, so that drug resistance against these two compounds is overcome in vitro . For the first time, we demonstrate in vitro efficacy of H. niger extract in cells of haematological malignancies and thus present an interesting baseline for further in vivo experiments.

Extracts of Helleborus species are used as phytopreparations with immunostimulatory properties in Romanian traditional medicine. In Germany, Helleborus niger is used in homeopathy and as an adjuvant therapy in the treatment of tumor patients in anthroposophical medicine. In vitro application of an aqueous extract from Helleborus niger resulted in a slight induction of sister chromatid exchanges (SCE) in cultured peripheral blood mononuclear cells (PBMC) from healthy individuals, an effect associated with a slight increase of the [3H]thymidine uptake in the DNA of isolated lymphocytes. Since the cytokines interleukin (IL)-2 and tumor necrosis factor (TNF)-α were reported to increase the number of SCE, we measured the concentrations of these cytokines in the supernatants of cultured PBMC treated with the plant extract. Here, no significant changes were observed as compared with the controls, but a trend to higher supernatant concentrations of TNF-α in six out of ten individuals was noted. Compared with lymphocytes treated with the alkylating substance, cyclophosphamide, the increase of the SCE levels induced by the plant extract is weak. The relevance of this DNA destabilizing property remains to be clarified.

Purpose Helleborus niger (Ranunculaceae), commonly known as Christmas rose, is used in anthroposophically extended cancer therapy in the adjuvant treatment of different entities and reduction of chemotherapy-associated side effects. Although Helleborus niger is widely used in anthroposophic medicine, there is a lack of scientific clinical and preclinical data and until now it is applied on an empirical basis. Neuroblastoma is one of the most common extracranial solid tumors of childhood, and more than 50 % of these children initially present with nonresectable primary tumors and disseminated metastasis to distant organ sites, predominantly bone marrow. In this study, we determined for the first time the cytotoxic properties of Helleborus niger Root (HNR) extract for neuroblastoma in vitro.

Helleborus niger, also known as Christmas Rose, belongs to the family of Ranunculaceae, a family of flowering plants with about 2500 different species. In complementary medicine Helleborus niger is used as adjuvant drug in the treatment of non-metastasised and metastasised forms of bronchial cancer, abdominal tumours and prostate cancer. It is also applied in myeloproliferative diseases like Hodgkin and Non-Hodgkin lymphoma, leukaemic disorders and AIDS- related diseases like the Kaposi sarcoma. Until now, there is no clinical or preclinical data regarding the effects of Helleborus niger in vivo, ex vivo or in vitro. For this purpose, we investigated the cytotoxic effects of four different standardized aqueous Helleborus niger extracts from the companies Hiscia and Helixor on various cancer cell lines. We used one whole plant extract, one root extract, one leave extract and one containing only the blossom of Helleborus niger. After 4h of treatment with the extracts no significant LDH release was measured, thus excluding an unspecific, necrotic damage of the cell membrane. After 24h a dose dependent inhibition of proliferation up to 69% could be found and after 48h a distinction into early (45,2%) and late apoptotic (45,5%) cells was detected via Annexin/PI staining. The cell cycle analysis revealed characteristic hypodiploid DNA fragments after 72h, once more identifying apoptosis as cause of the cell death. In the Western Blot analysis a processing of Caspase-3 could be found after 36 h incubation with the extract. Apoptotic cell death was detected in the Burkitt-like lymphoma cell line BJAB, the three human acute lymphoblastic leukemia cell lines NALM-6, Sup-B-15 and REH and the melanoma cell line MEL-HO. The apoptosis induction caused by the root extract was higher than the apoptotic cell death in the other extracts. There are two major pathways of apoptosis, the extrinsic pathway via death receptors like FADD and the intrinsic pathway via the mitochondria. In BJAB cells a breakdown of the mitochondrial membrane potential and dose-dependent mitochondrial permeability transition was detected after 48h, revealing that apoptosis is executed via the mitochondrial pathway. Furthermore, we found a decreased apoptosis induction in BCL-2 overexpressing melanoma cells. The dependency of Bcl-2 expression is another sign of apoptosis via the mitochondrial pathway. In contrast, apoptosis induction by Helleborus niger seems to be independent of Smac overexpression, which could be shown in Jurkat cells. In combination with the vinca alkaloid vincristine, which is used in the treatment of ALL, a synergistic effect could be detected. The apoptosis induction was up to 16% higher in combination than in the single treatment. Finally, we evaluated the effect on primary leukemia cells ex vivo. Interestingly, we could show a significant apoptosis induction in primary leukemia cells from 2 patients with ALL or AML in childhood, which were resistant to the treatment with the anthracycline doxorubicin. For the first time, we were able to show that extracts of Helleborus niger induce apoptosis in different cancer cell lines and primary leukemia cells. Apoptosis is executed via the intrinsic pathway and is independent of Smac overexpression. Thus, we present an interesting baseline for the design of upcoming in vivo experiments or clinical trials.

Methods currently available for evaluating pollen quality in vitro include, (a) tests of germinability; (b) tests of the stainability of the vegetative cell contents; (c) tests for enzyme activity, and (d) the fluorochromatic procedure (FCR), which tests principally the integrity of the plasmalemma of the vegetative cell. Using germinability in vitro as a standard, a comparison has been made between histochemical methods of classes (b), (c) and (d) in application to various pollens, immature, mature, and treated in ways known to affect viability and membrane state. Predictably, the lowest correlation was obtained with tests of stainability. The highest was given by the FCR, which generally provided an excellent guide to potential germinability. The FCR procedure is subject to various limitations, however, (a) A high correlation between FCR and germinability can only be expected when mature, ripe pollen is used; with immature pollen, the FCR will predict excessively high potential germinability. (b) The FCR may also predict a higher potential level of pollen function than in vitro germinability when the germination medium is sub-optimal. In this situation, however, it will generally give a better guide to fertilising capacity, (c) The FCR is not a test of pollen viability. Like germinability in vitro, it can yield a negative score with pollen which is nevertheless capable of functioning. For example, false negatives will be obtained with some species if the pollen is not properly pre-conditioned by rehydration before testing, an important point in monitoring stored pollen. The paper includes a brief discussion of the rationale of pollen testing.