Separation of bufadienolides from Helleborus thibetanus Franch. by a combination approach involving macroporous resin column chromatography and gradient countercurrent chromatography.

In this study, a combination approach involving macroporous resin (MR) column chromatography and gradient countercurrent chromatography (CCC) was employed to enrich and purify bufadienolides from the roots and rhizomes of Helleborus thibetanus Franch. Initially, a D101 MR-packed column chromatography was utilized for fractionation and enrichment of the bufadienolides, which were effectively eluted from the column using a 60% ethanol solution. CCC was subsequently introduced to separate the enriched product using the ethyl acetate/n-butanol/water (EBuWat, 4:1:5, v/v) and EBuWat (5:0:5, v/v) solvent systems in a gradient elution mode. As results, five bufadienolides, including 6.1 mg of hellebrigenin-3-O-ß-D-glucoside (1), 2.2 mg of tigencaoside A (2), 8.3 mg of deglucohellebrin (3), 3.5 mg of 14 ß-hydroxy-3ß-[ß-D-glucopyranosyl-(1→6)-(ß-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (4), and 3.0 mg of 14ß-hydroxy-3ß-[(ß-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (5), were effectively separated from 300 mg of the enriched product. The respective high-performance liquid chromatography purities were as follows: 95.2%, 75.8%, 85.7%, 82.3%, and 92.8%. This study provides valuable insights for the efficient enrichment and separation of bufadienolides from Helleborus thibetanus Franch.

Chemical constituents of Helleborus thibetanus / 中国中药杂志

The chemical constituents of Helleborus thibetanus were isolated and purified by silica gel column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative RP-HPLC, and the structures of all compounds were identified by modern spectrographic technology(MS, NMR). The MTT method was used to measure the cytotoxicity of compounds 1-8. Twelve compounds were isolated from the roots and rhizomes of H. thibetanus and were identified as(25R)-22β,25-expoxy-26-[(O-β-D-glucopyranosyl)oxy]-1β,3β-dihydroxyfurosta-5-en(1), β-sitosterol myristate(2), β-sitosterol lactate(3), β-sitosterol 3-O-β-D-glucopyrannoside(4), 4,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one(5), 1,3,5-trimethoxybenzene(6), 7,8-dimethylbenzo pteridine-2,4(1H,3H)-dione(7), 1H-indole-3-carboxylic acid(8), p-hydroxy cinnamic acid(9), lauric acid(10), n-butyl α-L-arabinofuranoside(11) and methyl-α-D-fructofuranoside(12), respectively. Among them, compound 1 is a new compound and named thibetanoside L; compounds 2, 5-8, 11 are first isolated from the family Ranunculaceae; compound 12 is isolated from the genus Helleborus for the first time. The results of MTT assay showed that the IC_(50) values of compounds 1-8 against HepG2 and HCT116 cells were greater than 100 μmol·L~(-1).

[Chemical constituents of Helleborus thibetanus].

The chemical constituents of Helleborus thibetanus were isolated and purified by silica gel column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative RP-HPLC, and the structures of all compounds were identified by modern spectrographic technology(MS, NMR). The MTT method was used to measure the cytotoxicity of compounds 1-8. Twelve compounds were isolated from the roots and rhizomes of H. thibetanus and were identified as(25R)-22ß,25-expoxy-26-[(O-ß-D-glucopyranosyl)oxy]-1ß,3ß-dihydroxyfurosta-5-en(1), ß-sitosterol myristate(2), ß-sitosterol lactate(3), ß-sitosterol 3-O-ß-D-glucopyrannoside(4), 4,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one(5), 1,3,5-trimethoxybenzene(6), 7,8-dimethylbenzo pteridine-2,4(1H,3H)-dione(7), 1H-indole-3-carboxylic acid(8), p-hydroxy cinnamic acid(9), lauric acid(10), n-butyl α-L-arabinofuranoside(11) and methyl-α-D-fructofuranoside(12), respectively. Among them, compound 1 is a new compound and named thibetanoside L; compounds 2, 5-8, 11 are first isolated from the family Ranunculaceae; compound 12 is isolated from the genus Helleborus for the first time. The results of MTT assay showed that the IC_(50) values of compounds 1-8 against HepG2 and HCT116 cells were greater than 100 µmol·L~(-1).

Chemical constituents isolated from the aerial parts of Helleborus cyclophyllus (A. Braun) Boiss. (Ranunculaceae), evaluation of their antioxidant and anti-inflammatory activity in vitro and virtual screening of molecular properties and bioactivity score.

Chemical investigation of ethyl acetate extract from the aerial parts of Helleborus cyclophyllus (A.Braun) Boiss. led to the isolation of ten natural products, and their structures were identified to be: 2-deoxy-D-ribono-1,4-lactone (1), 2-O-feruloyl-L-malate (2), three flavonoids: quercetin 3-O-ß-D-galactopyranoside (3), quercetin 3-O-6''-(3-hydroxy-3-methyl-gloutaryl)-ß-D-glucopyranoside (4) and quercetin 3-O-(2‴-caffeoylsophoroside) (5), 6-O-caffeoyl-1-O-p-coumaroyl-ß-D-glucopyranoside (6), two ecdysteroids: 20-hydroxyecdysone (7) and polypodine B (8) and two bufadienolides: deglucohellebrin (9) and hellebrin (10), on the basis of MS and NMR spectra. This is the first report on the occurrence of compounds (2)-(6) in the genus Helleborus. The chemotaxonomic significance of these compounds was summarised. Bioactivity score, molecular and pharmacokinetic properties of the isolated compounds were calculated by online computer software program Molinspiration. Compounds showed promising bioactivity scores for drug targets. Moreover, some of the isolated phenolic compounds were tested for their antioxidant and antiinflammatory activities. Tested compounds presented antioxidant ability correlated to the presence of the phenolic hydroxyl groups.

Bufadienolides and ecdysteroids from the whole plants of Helleborus niger and their cytotoxicity.

A new bufadienolide (1), two new bufadienolide glycosides (2 and 3), a new ecdysteroid (4), and four known compounds (5-8), were isolated from the whole plants of Helleborus niger L. (Ranunculaceae). The structures of the new compounds (1-4) were determined by spectroscopic analysis, including 2D NMR spectral data, and hydrolytic studies. Compounds 1-6 showed cytotoxicity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and SBC-3 human small-cell lung cancer cells, with IC50 values ranging from 0.0055 to 1.9 µM. HL-60 cells treated with either 3 or 4 showed apoptosis characteristics, such as nuclear chromatin condensation, accumulation of sub-G1 cells, and activation of caspase-3/7.

Antiseizure potential of the ancient Greek medicinal plant Helleborus odorus subsp. cyclophyllus and identification of its main active principles.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological data and ancient texts support the use of black hellebore (Helleborus odorus subsp. cyclophyllus, Ranunculaceae) for the management and treatment of epilepsy in ancient Greece. AIM OF THE STUDY: A pharmacological investigation of the root methanolic extract (RME) was conducted using the zebrafish epilepsy model to isolate and identify the compounds responsible for a potential antiseizure activity and to provide evidence of its historical use. In addition, a comprehensive metabolite profiling of this studied species was proposed. MATERIALS AND METHODS: The roots were extracted by solvents of increasing polarity and root decoction (RDE) was also prepared. The extracts were evaluated for antiseizure activity using a larval zebrafish epilepsy model with pentylenetetrazole (PTZ)-induced seizures. The RME exhibited the highest antiseizure activity and was therefore selected for bioactivity-guided fractionation. Isolated compounds were fully characterized by NMR and high-resolution tandem mass spectrometry (HRMS/MS). The UHPLC-HRMS/MS analyses of the RME and RDE were used for dereplication and metabolite profiling. RESULTS: The RME showed 80% inhibition of PTZ-induced locomotor activity (300 µg/ml). This extract was fractionated and resulted in the isolation of a new glucopyranosyl-deoxyribonolactone (1) and a new furostanol saponin derivative (2), as well as of 20-hydroxyecdysone (3), hellebrin (4), a spirostanol glycoside derivative (5) and deglucohellebrin (6). The antiseizure activity of RME was found to be mainly due to the new furostanol saponin (2) and hellebrin (4), which reduced 45% and 60% of PTZ-induced seizures (135 µM, respectively). Besides, the aglycone of hellebrin, hellebrigenin (S34), was also active (45% at 7 µM). To further characterize the chemical composition of both RME and RDE, 30 compounds (A7-33, A35-37) were annotated based on UHPLC-HRMS/MS metabolite profiling. This revealed the presence of additional bufadienolides, furostanols, and evidenced alkaloids. CONCLUSIONS: This study is the first to identify the molecular basis of the ethnopharmacological use of black hellebore for the treatment of epilepsy. This was achieved using a microscale zebrafish epilepsy model to rapidly quantify in vivo antiseizure activity. The UHPLC-HRMS/MS profiling revealed the chemical diversity of the extracts and the presence of numerous bufadienolides, furostanols and ecdysteroids, also present in the decoction.

Novel Steroidal Glycosides from the Whole Plants of Helleborus foetidus.

Phytochemical analysis of the whole Helleborus foetidus plants identified 28 steroidal glycosides (1-28), including 20 novel spirostanol glycosides (1-20) and a novel furostanol glycoside (21). The structures of the newly identified compounds were elucidated by two-dimensional NMR spectroscopy and hydrolytic cleavage. Compounds 12, 13, and 15 were determined to be spirostanol trisdesmosides bearing sugar moieties at the C-1, -21, and -24 hydroxy groups of the aglycone unit. The isolated compounds were subsequently evaluated for cytotoxic activity against HL-60 human promyelocytic leukemia cells and A549 human lung carcinoma cells. In particular, 7 showed cytotoxic activity against the HL-60 and A549 cells, with IC50 values of 5.9 and 6.6 µM, respectively, whereas 19 was selectively cytotoxic to A549 cells with an IC50 value of 5.5 µM.

New cytotoxic bufadienolides from the roots and rhizomes of Helleborus thibetanus Franch.

Three new bufadienolides 14ß, 16ß-dihydroxy-3ß-[ß-D-glucopyranosyl-(1→6)-(ß-D-glucopyranosyl)oxy]-5α-bufa-20, 22-dienolide (1), 14ß-hydroxy-3ß-[ß-D-glucopyranosyl-(1→4)-(ß-D-glucopyranosyl)oxy]-5α-bufa-20, 22-dienolide (2) and hellebrigenin-3-O-ß-D-glucosyl-(1→4)-ß-D-glucoside (3), together with eight known bufadienolides (4-11) were isolated from the roots and rhizomes of Helleborus thibetanus. Their structures were elucidated by extensive spectroscopic methods and acid hydrolysis. Compounds 1-7 were evaluated for their cytotoxic activity against HCT116, A549 and HepG2 tumor cell lines. Compound 1 exhibited moderate cytotoxicity against HepG2 cells with IC50 value of 15.1 ± 1.72 µM. Compounds 5 and 6 exhibited moderate cytotoxicity against HCT116 cells with IC50 values of 15.12 ± 0.58 µM and 13.17 ± 2.34 µM, respectively.

Three new spirostanol glycosides from Helleborus thibetanus.

An ongoing chemical investigation on n-BuOH extract of roots and rhizomes of Helleborus thibetanus afforded three new spirostanol glycosides (1-3). Their structures were elucidated by extensive analysis of 1 D, 2 D NMR spectra, together with IR and MS methods and acid hydrolysis. This is the first report of the isolation of spirostanol glycoside with xylose at C-24 of the aglycone in Helleborus.

Studies on the constituents of Helleborus purpurascens: use of derivatives from calix[6]arene, homooxacalix[3]arene and homoazacalix[3]arene as extractant agents for amino acids from the aqueous extract.

The task of this work was to investigate the extraction capacity of various calixarenes for free and esterified amino acids from aqueous acid phases. Furthermore, this method was applied to aqueous extracts of Helleborus purpurascens. Generally, it is known that calixarenes can be used as extractants for ammonium compounds due to π-cation and lone pair cation interactions. As first, tert-Butyl-calix[6]arene and derivatives thereof were used. They had already proven their worth in previous investigations. In addition, tert-Butyl-hexahomooxa-calix[3]arene was used also, which can also enter into lone pair cation interactions. In addition to these well-known calixarenes, new calixarenes were produced and tested. Based on the tert-Butyl-hexahomooxa-calix[3]arene, a phosphor(III)bridged derivative was prepared, combining the three aromatic hydroxyl groups to a phosphite. As a seldom-described class of calixarenes, tert-Butyl-hexahomoaza-calix[3]arene derivatives were used. The nitrogen analogues of tert-Butyl-hexahomooxa-calix[3]arene could be produced as N-benzyl derivatives. The structure of the esterified carboxymethylated derivative of N,N',N″-Tribenzyl-tert-Butyl-hexahomoaza-calix[3]arene could be verified by X-ray structure analysis. It crystallized as a partial cone. The extraction capacity of the described calixarenes was investigated for amino acids from aqueous acidic solutions into an organic phase. For the testing were chosen asparagine, aspartic acid, tyrosine, tryptophane, phenylalanine and pipecolinic acid and their methyl esters. The amino acids and their methyl esters were dissolved in water at different pH values. The calixarenes were dissolved in dichloromethane (DCM) or chloroform. After this preparation, the aqueous acidic amino acid solutions were mixed with the solutions and shaken intensively. In addition, blank values were determined by extracting the aqueous stock solutions of the amino acids and their methyl esters with pure solvents. To determine the extraction rate, the phases were separated and each analysed using GC-FID, partially GC-MS(EI). The evaluation is performed in two ways. On the one hand the depletion in the aqueous phase and on the other hand the content in the organic phase was determined.

Cardiac Glycoside Compound Isolated from Helleborus thibetanus Franch Displays Potent Toxicity against HeLa Cervical Carcinoma Cells through ROS-Independent Autophagy.

The current study aimed to examine the anticancer activity of HTF-1, a cardiac glycoside (CG) isolated from Helleborus thibetanus Franch, using a cell-based model and to discover the underlying mechanisms with specific focus on autophagy. We found that HTF-1 was able to potently decrease the viability of several cancer cell lines especially for HeLa cervical carcinoma cells. It was discovered that HTF-1 dose dependently induced overproduction of ROS in HeLa cells, and the cell viability can be rescued when adding ROS scavenger N-acetyl-l-cysteine (NAC). More, we found that HTF-1 induced ROS-independent autophagy in concentration- and time-dependent manners in HeLa cells. This can be collectively verified by LC3-II and p62 abundance and also eGFP-LC3 puncta assay, bafilomycin clamp experiment, and acidotropic dye fluorescent labeling experiment. Additionally, TEM examination showed more autophagic vacuoles for HTF-1-treated HeLa cells. In HeLa cells, pretreatment with wortmannin (an inhibitor of the initial stages of autophagy to block autophagosome formation, thus, it should weaken the autophagy induction effect of HTF-1) decreased the autophagic flux and partially antagonized cell death induced by HTF-1, indicating that autophagy induced by HTF-1 played a cancer-suppressing role. Furthermore, coadministration of BAF (as a distal inhibitor of autophagy) with HTF-1 demonstrated a synergistic anticancer effect against HeLa cells. We believe that our work will enrich the understanding of CGs and especially anticarcinoma activity, also, pave the way for natural-product-based anticancer drug development.

Steroidal constituents from Helleborus thibetanus and their cytotoxicities.

Thibetanosides E-H (1-4), four new steroidal constituents including three rare sulfonates (2-4), were isolated from the roots and rhizomes of Helleborus thibetanus, together with nine known steroidal compounds (5-13). Their structures were elucidated by detailed spectroscopic analysis, including 1D and 2D NMR techniques and chemical evidence. In this study, compounds 2-13 were evaluated for their cytotoxic activities against HCT116, A549 and HepG2 tumor cell lines in vitro. Among them, compound 8 (thibetanoside C) showed cytotoxicities against A549 cells(IC50 39.6 ± 1.9 µmol·L-1) and HepG2 cells(IC50 41.5 ± 1.1 µmol·L-1), respectively. Compound 9 (23S, 24S)-24-[(O-ß-D-fucopyranosyl)oxy]-3ß, 23-dihydroxy-spirosta-5, 25(27)-diene-1ß-ylO-(4-O-acetyl- α-L-rhamnopyranosyl)-(1→2)-O-[ß-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside) showed cytotoxicity against HCT116 cells(IC50 33.6 ± 2.1 µmol·L-1).

Preclinical evaluation of safety and potential of black hellebore extracts for cancer treatment.

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.

A cardiac glycoside HTF-1 isolated from Helleborus thibetanus Franch displays potent in vitro anti-cancer activity via caspase-9, MAPK and PI3K-Akt-mTOR pathways.

Experiments have been undertaken and for the first time, we have identified that a new cardiac glycoside (CG) isolated from Helleborus thibetanus Franch. a plant endemic to China, bears potent anti-cancer activity. We have named it as HTF-1. By using in vitro cell models, we have found that HTF-1 induces apoptosis against several types of cancer cells in a concentration- and time-dependent manner. It is able to inhibit cancer cell in proliferation, migration and invasion. HTF-1 causes S cell cycle arrest. Further-on, we have identified that HTF-1 triggers caspase-9 dependent apoptosis pathway and double strand DNA breaks (DSBs). Additionally, HTF-1 activates JNK, but suppresses ERK and PI3K-Akt-mTOR pathways. Collectively, the above-mentioned mechanisms contribute to the anti-cancer activity of HTF-1. It is rare to discover novel anti-cancer CG during the past couple of decades. We believe that our work will enrich the understanding of CGs; also, pave the way for natural product-based anti-cancer drug development.

Steroids from Helleborus caucasicus reduce cancer cell viability inducing apoptosis and GRP78 down-regulation.

Helleborus caucasicus (Ranunculaceae) is an endemic plant of the Caucasian flora, widely distributed in West Georgia. Biological activities for the extracts of some Helleborus species including H. caucasicus have been reported. In this work we found that butanolic extract of the underground parts of H. caucasicus and isolated compounds decreased cell viability in vitro on cancer cell line of lung origin (Calu-1) in a concentration-dependent manner, compared to the normal cell line. In particular, we identified that furostanol derivative (25S)-22α,25-epoxyfurost-5-ene-3ß,11ß,26-triol 26-O-ß-d-glucopyranoside (5), 20-hydroxyecdysone (6), and 3ß,5ß,14ß-trihydroxy-19-oxo-bufa-20,22-dienolide 3-O-α-l-rhamnopyranoside, known as deglucohellebrin (7) exerted a strong cytotoxic effect on the same cells and on other cancer cell lines (HepG2 and Caco-2) reducing the S-phase entry (compound 6) and inducing cell apoptosis associated with activation of caspase-3 (compound 7). Moreover we demonstrated that 6 and 7 significantly decreased protein expression of GRP78, a general ER-stress marker, suggesting pro-apoptotic functions. These findings indicated that selected compounds from H. caucasicus are potential interesting agents in anti-cancer therapy.

Studies on the constituents of Helleborus purpurascens: analysis and biological activity of the aqueous and organic extracts.

In Southeast Europe, the ethnomedicinal use of Helleborus species has a very long tradition. Cardiac steroids (Hellebrin), cysteine-rich proteins (Hellethionins) and several steroidal saponins have been identified in these plants. Aim of the present work was to investigate the amino acid composition of native extracts from the root and rootstock of Helleborus purpurascens. The amino acids have been identified by the GC-MS technique on the previously derivatised (Phenomenex Faast Kit) extract samples by comparison with the mass spectra and retention-time of the standards. A remarkable finding was a relatively intensive peak attributed to the non-proteinogenic Pipecolic acid (Pic). A cyclisation of the derivatised glutamine was observed during the GC measurement and a mechanistic pathway is described. Samples of the extract and of some isolated fractions have also been tested on; altogether 12 cancer cell lines aimed to identify further potentially cytostatic components which should be less toxic than Hellebrin. The finding of one Hellebrin-free fraction (IC50 = 0.007 mg/L) with higher cytotoxicity than Hellebrin (IC50 = 0.008 mg/L) is remarkable.

New minor spirostanol glycosides from Helleborus thibetanus.

Phytochemical reinvestigation of the dried roots and rhizomes of Helleborus thibetanus afforded four new minor spirostanol glycosides (1-4) and four known spirostanol glycosides (5-8). Their structures were determined on the basis of spectroscopic analyses, including 1D and 2D NMR experiments, together with HR-ESI-MS and IR measurements and the results of acid hydrolysis.

Three new steroidal saponins from Helleborus thibetanus.

Three new steroidal saponins including two spirostanol glycosides (1-2) and one furostanol glycoside 1-sulphate (3) were isolated from the dried roots and rhizomes of Helleborus thibetanus. Structures of the compounds were determined on the basis of extensive use of 1-D and 2-D NMR experiments, together with HR-ESI-MS and IR measurements, as well as the results of acid hydrolysis. Compounds 1-2 represented steroidal saponins with an unusual substitution pattern, which possessed a double bond at C-25 and were glycosylated at 1-OH.

Helleborus purpurascens-Amino Acid and Peptide Analysis Linked to the Chemical and Antiproliferative Properties of the Extracted Compounds.

There is a strong drive worldwide to discover and exploit the therapeutic potential of a large variety of plants. In this work, an alcoholic extract of Helleborus purpurascens (family Ranunculaceae) was investigated for the identification of amino acids and peptides with putative antiproliferative effects. In our work, a separation strategy was developed using solvents of different polarity in order to obtain active compounds. Biochemical components were characterized through spectroscopic (mass spectroscopy) and chromatographic techniques (RP-HPLC and GC-MS). The biological activity of the obtained fractions was investigated in terms of their antiproliferative effects on HeLa cells. Through this study, we report an efficient separation of bioactive compounds (amino acids and peptides) from a plant extract dependent on solvent polarity, affording fractions with unaffected antiproliferative activities. Moreover, the two biologically tested fractions exerted a major antiproliferative effect, thereby suggesting potential anticancer therapeutic activity.

LC-MS(n) characterization of steroidal saponins in Helleborus niger L. roots and their conversion products during fermentation.

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 ß-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, ß-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.