“…Gradient elution was carried out for both analyte groups, and detection wavelengths were 210 nm (saponins) and 261 nm (protoanemonin). Further details on both HPLC methods as well as method validation data have been reported earlier [21]. Fig.…”
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.
“…Gradient elution was carried out for both analyte groups, and detection wavelengths were 210 nm (saponins) and 261 nm (protoanemonin). Further details on both HPLC methods as well as method validation data have been reported earlier [21]. Fig.…”
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.
“…The results revealed that H. niger has a concentration-dependent cytotoxic and antiproliferative effect on all lines used [20]. In addition, Schink and colleagues highlighted the cytotoxic effect of H. niger extract on the melanoma cell line due to its ability to induce cell apoptosis [21]. Other species such as H. odorus, H. multifidus, and H. hercegovinus have been tested in vitro on Burkitt's lymphoma B cells (BJAB).…”
In the field of oncology, the plant kingdom has an inexhaustible supply of bioactive compounds. Phytochemical compounds isolated from Helleborus species have been found to be useful in various chronic diseases. This has brought Helleborus to the attention of medical researchers. H. purpurascens is a plant characteristic of the Carpathian area, known since ancient times for its beneficial effects. The aim of the study was to evaluate the flavonoids composition of a hydroalcoholic extract of H. purpurascens, as well as to assess its antioxidant activity and antitumor potential at the level of two healthy cell lines and four tumor cell lines. In addition, the expression of the genes involved in the apoptotic process (Bcl-2, Bad, and Bax) were evaluated. The results indicated that the extract has a high concentration of flavonoids, such as epicatechin, quercetin, and kaempferol. The extract has an increased antioxidant activity, very similar to that of the standard, ascorbic acid and cytotoxic effects predominantly in the breast cancer cell line, being free of cytotoxic effects in healthy cell lines. Underlying the cytotoxic effect is the induction of the process of apoptosis, which in the present study was highlighted by decreasing the expression of anti-apoptotic genes (Bcl-2) and increasing the expression of pro-apoptotic genes (Bad and Bax). In conclusion, the hydroalcoholic extract of H. purpurascens can be considered an important source for future medical applications in cancer therapy.
“…Dose-response assays (Figure 1) were performed by means of the Hoechst 33342/PI dual staining method and results indicated that A. nemorosa inhibits proliferation of HeLa cells in a dose dependent manner. For a crude extract to be considered promising for systemic application, the IC 50 should be less than 100 µg/mL in vitro [16]. The IC 50 of A. nemorosa was determined to be 20.33 ± 2.480 µg/mL.…”
Anemone nemorosa is part of the Ranunculaceae genus Anemone (order Ranunculales) which comprises more than 150 species. Various parts of the plant have been used for the treatment of numerous medical conditions such as headaches, tertian agues, rheumatic gout, leprosy, lethargy, eye inflammation as well as malignant and corroding ulcers. The Anemone plants have been found to contain various medicinal compounds with anti-cancer, immunomodulatory, anti-inflammatory, anti-oxidant and anti-microbial activities. To date there has been no reported evidence of its use in the treatment of cancer. However, due to the reported abundance of saponins which usually exert anti-cancer activity via cell cycle arrest and the induction of apoptosis, we investigated the mode of cell death induced by an aqueous A. nemorosa extract by using HeLa cervical cancer cells. Cisplatin was used as a positive control. With a 50% inhibitory concentration (IC50) of 20.33 ± 2.480 µg/mL, treatment with A. nemorosa yielded a delay in the early mitosis phase of the cell cycle. Apoptosis was confirmed through fluorescent staining with annexin V-FITC. Apoptosis was more evident with A. nemorosa treatment compared to the positive control after 24 and 48 h. Tetramethylrhodamine ethyl ester staining showed a decrease in mitochondrial membrane potential at 24 and 48 h. The results obtained imply that A. nemorosa may have potential anti-proliferative properties.
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