Effect of extraction, microbial fermentation and storage on the cardenolide profile of Strophanthus kombé Oliv. seed preparations.

Strophanthus extracts containing cardioactive cardenolides are still applied in European complementary medicine for the treatment of heart diseases. However, the cardenolide profile and the fate of individual compounds during extraction and storage are not well understood. Therefore, the objective of the present study was to characterize the cardenolide compound pattern in extracts of different polarity and their structural changes upon storage in aqueous fermented preparations. For this purpose, individual cardenolides were quantitated by a UHPLC-DAD validated method using an internal standard. Three different extraction protocols were compared: hydroethanolic extraction under reflux with and without previous defatting of the seed material and ultrasonic-assisted extraction at ambient temperature. Reflux extraction of non-defatted seeds showed maximum cardenolide yields. Differences in the cardenolide contents of seeds of the different origins Zimbabwe and Malawi were observed. The cardenolide profile and metabolization of individual compounds upon fermentation and storage of S. kombé seed extracts revealed that predominant cardenolides, mainly strophanthidin glycosides, changed upon storage over 12 months. Cardenolides exhibiting two or three saccharide moieties were degraded presumably by ß-glucosidase activities, originating from the plant material or lactobacilli, releasing the corresponding monoglycosides. The latter were further degraded into the corresponding aglycones probably by acid hydrolysis as a result of lactic acid accumulation.

1-Acetyl-3-[(3R)-hydroxyfatty acyl]glycerols: Lipid Compounds from Euphrasia rostkoviana Hayne and E. tetraquetra (Bréb.) Arrond.

Five homologous acetylated acylglycerols of 3-hydroxyfatty acids (chain lengths C(14) - C(18)), named euphrasianins A - E, were characterized for the first time in Euphrasia rostkoviana Hayne (Orobanchaceae) by gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (HPLC/APCI-MS(n) ). In addition to mass spectrometric data, structures of euphrasianins were verified via a three-step total synthesis of one representative homologue (euphrasianin A). The structure of the latter was confirmed by 1D- and 2D-NMR experiments as well as high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS). The absolute configuration of the 3-hydroxyfatty acid moiety at C(3) was found to be R in the natural euphrasianins, which was determined by alkaline hydrolysis and methylation of a purified fraction, followed by chiral GC analysis. Furthermore, in extracts of Euphrasia tetraquetra (Bréb.) Arrond. euphrasianins C and E were detected exclusively, indicating that this subclass of lipid constituents is possibly valuable for fingerprinting methods.

Characterization of the cardiac glycoside and lipid profiles of Strophanthus kombé Oliv. seeds.

The seeds of Strophanthus kombé Oliv. are known to contain high levels of cardioactive compounds. However, the therapeutic use of Strophanthus in the treatment of cardiopathy requires more detailed knowledge of the compound profile to profit from the full potential of Strophanthus preparations. Therefore, the objective was to characterize the cardenolide profile and lipophilic constituents in S. kombé seeds using methods applicable in routine quality control. Freshly prepared S. kombé seed extracts were analyzed without previous sample clean-up using a novel HPLC-DAD-MSn method. In addition, seed oils were analyzed by GC-MS following derivatization of the lipids. More than 20 cardenolides were tentatively assigned in the seed extracts including strophanthidin, strophanthidol, periplogenin and strophanthidinic acid aglycones, carrying various saccharide moieties. The findings revealed the presence of eight novel cardenolides, which have not been described for S. kombé so far. The occurrence of strophanthidinic acid derivatives was verified by comparison with synthesized strophanthidinic acid-cymaropyranoside. GC-MS characterization of the oils mainly revealed the presence of fatty acids, especially oleic acid and linoleic acid, as well as phytosterols, the latter representing intermediates of cardenolide biosynthesis. In summary, these findings broaden our knowledge on the secondary metabolism of Strophanthus.

Metabolic fate of cardiac glycosides and flavonoids upon fermentation of aqueous sea squill (Drimia maritima L.) extracts.

Sea squill (Drimia maritima L.) extracts have been used for centuries for the medical treatment of heart diseases. A procedure for the preparation of Drimia extracts applied for such purposes comprising a fermentation step is described in the German Homoeopathic Pharmacopoeia (GHP). However, little is known about the secondary metabolite profile of such extracts and the fate of these components upon processing and storage. Thus, in the present study sea squill extracts were monitored during fermentation and storage by HPLC-DAD-MS(n) and GC-MS to characterise and quantitate individual cardiac glycosides and phenolic compounds. For this purpose, a previously established HPLC method for the separation and quantitation of pharmacologically relevant cardiac glycosides (bufadienolides) was validated. Within 12 months of storage, total bufadienolide contents decreased by about 50%, which was attributed to microbial and plant enzyme activities. The metabolisation and degradation rates of individual bufadienolide glycosides significantly differed, which was attributed to differing structures of the aglycones. Further degradation of bufadienolide aglycones was also observed. Besides reactions well known from human metabolism studies, dehydration of individual compounds was monitored. Quantitatively predominating flavonoids were also metabolised throughout the fermentation process. The present study provides valuable information about the profile and stability of individual cardiac glycosides and phenolic compounds in fermented Drimia extracts prepared for medical applications, and expands the knowledge of cardiac glycoside conversion upon microbial fermentation.

Simultaneous determination of bufadienolides and phenolic compounds in sea squill (Drimia maritima (L.) Stearn) by HPLC-DAD-MSn as a means to differentiate individual plant parts and developmental stages.

Mediterranean sea squill (Drimia maritima (L.) Stearn) is used in the production of medicinal products. Current HPLC methods comprise tedious sample clean-up and have been merely focused on the analysis of cardiac glycosides, whereas a thorough characterization of D. maritima considering both the latter compound class and more hydrophilic secondary metabolites in one HPLC run has not been performed so far. Consequently, a novel HPLC-DAD-MS(n) method has been developed allowing the simultaneous determination of both cardiac glycosides and phenolic compounds, which is characterized by simplified sample preparation. This method was applied to characterize sea squill, revealing a complex profile of its extractive compounds derived from the two classes. Furthermore, the potential of the method reported here to quantitate the predominant compounds, i.e., dihydroquercetin derivatives and bufadienolides, was demonstrated. The occurrence of phenolic compounds, not described for sea squill so far, and of characteristic compounds specific to individual plant parts or vegetation stages was further addressed. The data revealed that classification of various vegetation phases based on quantitative evaluation of bufadienolides and dihydroquercetin derivatives applying principal component analysis (PCA) appears possible. Thus, the methodology presented here forms the basis for future routine application in quality control of raw materials and pharmaceutical preparations derived from sea squill. This will allow systematic comparison of different plant parts, vegetation stages and origins based on an extended sample set.