Abstract: INTRODUCTION: Pancratium canariense Ker Gawler is a plant species belonging to family Amaryllidaceae. Plants from this family are known to synthesise a particular type of bioactive compounds, named Amaryllidaceae alkaloids, which have shown AChE inhibitory activity. OBJECTIVE: To perform the metabolite profiling of methanolic extracts from P. canariense in order to identify bioactive compounds. METHODOLOGY: Methanolic extracts from bulbs, leaves and fruits were separated into alkaloid-free apolar and polar fractions, as well as alkaloid fractions, and subjected to AChE assay. Metabolite profiling of extracts and fractions of P. canariense was carried out by GC-EI-MS and LC-ESI-TOF-MS. RESULTS: AChE inhibitory activities of the alkaloid fractions at a concentration of 10 microg/mL were 29.80 +/- 0.91, 40.93 +/- 4.60 and 58.06 +/- 1.18% for the bulbs, leaves and fruits, respectively. Seventy-six metabolites-mono-, di- and trisaccharides, fatty acids, amino acids, sterols as well as several Amaryllidaceae alkaloids-were detected. Further purification of the alkaloids from the methanolic extracts resulted in the detection of 31 compounds including several potent AChE inhibitors such as habranthine and galanthamine, and the structural elucidation of 3-O-acetylhabranthine, a new natural compound with potential AChE inhibitory activity. CONCLUSION: The described method resulted in effective integration of both GC-EI-MS and LC-ESI-TOF-MS strategies, which permitted the identification of many metabolites, as well as the structural elucidation of new compounds with potential AChE inhibitory activity.
Abstract: Phytochemical studies on Galanthus species resulted in the isolation of three new compounds: 3,3'-O-(3',3''-dihydroxybutanoyl)hamayne and 11,3'-O-(3',3''-dihydroxybutanoyl)hamayne from G. nivalis and 2-O-(3'-hydroxybutanoyl)lycorine from G. elwesii. Additionally, 3,11-O-(3',3''-dihydroxybutanoyl)hamayne, 3,11,3'-O-(3',3'',3'''-trihydroxybutanoyl)hamayne, 8-O-demethylvasconine, tazettine, epimacronine, and ismine from G. nivalis; 2-O-(3'-acetoxybutanoyl)lycorine and incartine from G. elwesii; and hamayne, 11-O-(3'-hydroxybutanoyl)hamayne and lycorine from both species were isolated. Their structures were determined by EI-MS, HR-MS, CD, and 1D and 2D NMR (COSY, NOESY, HMQC, and HMBC) experiments.
Abstract: The alkaloids of intact plants, calli and shoot-clump cultures of L. aestivum were analyzed by GC-MS. Twenty-four alkaloids were detected. Calli appeared to produce sparse alkaloid profiles in stark contrast to shoot-clumps that had similar profiles to those of the intact plant. Seven shoot-clump strains produced galanthamine predominantly whereas another three were dominated by lycorine. Shoot-clump strains cultivated under light accumulated about two-times more galanthamine (an average of 74 microg/g of dry weight) than those cultivated in darkness (an average of 39 microg/g of dry weight). In comparison to intact plants, the shoot-clumps accumulated 5-times less galanthamine. The high variability of both the galanthamine content (67% and 75% of coefficient of variation under light and darkness conditions, respectively) and alkaloid patterns indicates that the shoot-clump cultures initiated from callus could be used as a tool for improvement of the in vitro cultures.
Abstract: Hyoscyamine biosynthesis in Datura stramonium hairy roots with different ploidy levels was investigated. For the first time, we report that hairy roots undergo endoreduplication and therefore consist mainly of cells with doupled sets of chromosomes of primary plant tissues, used for Agrobacterium transformation. The alkaloid profiles of hairy roots obtained from diploid and tetraploid plants were similar in terms of the major compounds, but they differed significantly with respect to the minor compounds (here defined as those that accounted for <1% of the total ion current of the alkaloid mixture in gas chromatography-mass spectrometric analyses). Significant differences in the effects of the main nutrients on the growth of the hairy roots obtained from diploid and tetraploid plants and their hyoscyamine contents were observed. The maximal yield of hyoscyamine (177 mg/L) was obtained when hairy roots from tetraploid plants were cultivated in Murashige-Skoog nutrient medium supplemented with 6% sucrose. Time courses of utilization of the main nutrients in the medium during cultivation of D. stramonium hairy root cultures are also presented.
Abstract: The common effect of NH4+, NO3-, KH2PO4 and sucrose on the biosynthesis of galanthamine by a Leucojum aestivum shoot culture was studied. Polynominal regression models were elaborated for the description of the galanthamine biosynthesis as a consequence of variation of the investigated variables (NH4+ between 0.20 and 0.54 g/L; NO3- between 1.44 and 3.44 g/L; KH2PO4 between 0.10 and 0.24 g/L, and sucrose between 30.00 and 60.00 g/L). Optimization procedures allowed us to establish the optimal concentrations of the investigated variables and to propose the modified MS nutrient medium, with 4.50 g/L KNO3, 0.89 g/L NH4NO3, 1.25 g/L (NH4)2SO4, 0.10 g/L KH2PO4 and 60 g/L sucrose, for the galanthamine production by a Leucojum aestivum shoot culture. The proposed modified MS medium provided considerable increase of both the production yield and the relative content of the target alkaloid in the alkaloid mixture.
Abstract: Twenty-one alkaloids and related compounds were found in Sternbergia colchiciflora (Amaryllidaceae), a hitherto not studied plant species. Twenty of them were detected by GC-MS in the crude extracts of this plant species. Ten alkaloids were isolated and their structures confirmed by NMR, MS and CD measurements. Many of the compounds found in this species, such as lycorine, tazettine, haemanthidine, are known to possess strong bioactivity. Variations in the alkaloid pattern were found during the phenological cycle of the plant. Lycorine-type compounds were dominant in the plant organs during both the flowering period and dormancy. The alkaloid pattern during both periods of leaf development and fructification was dominated by haemanthamine-type in the leaves and lycorine-type compounds in the bulbs, respectively.
Abstract: N-(14-Methylallyl)norgalanthamine, a new natural compound, together with five known alkaloids: N-allylnorgalanthamine, galanthamine, epinorgalanthamine, narwedine, and lycorine were isolated from mother liquors (waste material) obtained after industrial production of galanthamine hydrobromide from Leucojum aestivum leaves. The structures of N-allylnorgalanthamine and N-(14-methylallyl)norgalanthamine were completely determined by (1)H and (13)C NMR spectroscopy and two-dimensional experiments. N-allylnorgalanthamine (IC(50)=0.18microM) and N-(14-methylallyl)norgalanthamine (IC(50)=0.16microM) inhibit AChE considerably more than the approved drug galanthamine (IC(50)=1.82microM).
Abstract: Galanthamine, an acetylcholinesterase inhibitor used for the treatment of Alzheimer's disease, and galanthamine-type alkaloids are synthesised in different plants of the family Amaryllidaceae. A capillary gas chromatographic-mass spectroscopic (CGC-MS) method for the separation of 7 galanthamine type alkaloids, including galanthamine and epigalanthamine, is described in the present paper. A simple method for the routine quantification of galanthamine in plants was developed using pre-packed columns with diatomaceous earth (Isolute HM-N), allowing simultaneous preparation of a large number of samples. Galanthamine showed excellent linearity in the range from 50 to 1000 microg/mL and the limit of quantification was 5 microg/mL in total ion current mode and 1.6 ng/mL in selected ion monitoring mode. The recovery of galanthamine was more than 90%. Interday reproducibility (RSD) of the extraction was 2.74%. A method to find and to microextract Amaryllidaceae alkaloids in low-mass plant samples is also described.
Abstract: Alkaloid extracts from 12 plant species of the families Amaryllidaceae, Fumariacae and Papaveraceae were studied with respect to their acetylcholinesterase inhibitory activity and alkaloid patterns. Fifty-three alkaloids were identified by GC-MS, including known acetylcholinesterase (AChE) inhibitors such as galanthamine, epigalanthamine, sanguinine and epinorgalanthamine in extracts of Amaryllidaceae plants and protopine in extracts of Fumariaceae and Papaveraceae plants. The galanthamine-containing extracts of the amaryllidaceous plants were found to be the most active while the extract of Corydalis bulbosa was the most active among the extracts of the tested plants from the Fumariaceae and Papaveraceae plants. TLC bioautographic assay, preparative TLC and GC-MS analysis were combined to identify the active compounds in the studied extracts. Galanthamine was isolated from the known AChE inhibitors in the extracts of Amaryllidaceae plants. Corydaline, bulbocapnine and stylopine were found to be active in the extracts of plant species of the families Fumariaceae and Papaveraceae. Available standards of deshydrocorydaline--a precursor of corydaline, corydaline and stylopine--were tested for AChE inhibitory activity. Deshydrocorydaline and corydaline showed potent inhibitory activity comparable with that of the positive control galanthamine.
Abstract: Phytochemical studies on Galanthus elwesii resulted in the isolation of five alkaloids: incartine, hordenine, hippeastrine, 8-O-demethylhomolycorine and lycorine. The NMR data given previously for incartine were revised and completed by two-dimensional 1H-1H and 1H-13C chemical shift correlation experiments. In vitro studies on the bioactivity of incartine were carried out.
Abstract: Phytochemical studies on Galanthus nivalis of Bulgarian origin resulted in the isolation of five compounds: 11-O-(3'-hydroxybutanoyl)hamayne, 3,11-O-(3',3''-dihydroxybutanoyl)hamayne, 3-O-(2''-butenoyl)-11-O-(3'-hydroxybutanoyl)hamayne, 3,11,3''-O-(3',3'',3'''-trihydroxybutanoyl)hamayne, and 2-O-(3'-acetoxybutanoyl)lycorine, together with five known alkaloids: ungeremine, lycorine, tazettine, hamayne, and ismine. Their structures were determined by (1)H and (13)C NMR spectroscopy and two-dimensional (1)H-(1)H and (1)H-(13)C chemical shift correlation experiments.
Abstract: Leucojum aestivum (summer snowflake) is a plant species used for the extraction of galanthamine, an acetylcholinesterase inhibitor for the treatment of Alzheimer's disease. Extracts from bulbs collected from 18 Bulgarian populations and from shoot-clumps obtained in vitro from 8 different populations showed variations in their alkaloid composition. Nineteen alkaloids were detected in the studied samples by GC-MS. Typically, the alkaloid fractions of L. aestivum bulbs were dominated by galanthamine type compounds, but lycorine, haemanthamine and homolycorine type alkaloids were also found as dominant compounds in some of the samples. Extracts from the shoot-clumps obtained in vitro were found to contain galanthamine or lycorine as main alkaloids. The galanthamine content ranged from 28 to 2104 microg/g dry weight in the bulbs, and from traces to 454 microg/g dry weight in the shoot-clumps.
Abstract: A comparative GC-MS investigation of the alkaloid patterns of three varieties of Datura stramonium vars. stramonium, tatula and godronii, was carried out. Twenty-five tropane alkaloids were identified in the plant organs. Alkaloid patterns of the roots, leaves and seeds of the varieties grown at equal conditions in Bulgaria were very similar. In contrast, alkaloid pattern of D. stramonium var. stramonium, grown in Egypt, showed significant differences indicating that it is influenced more strongly by the environmental factors than genetic ones.
Abstract: Underivatised alkaloid mixtures extracted from intact plants and in vitro cultures of Leucojum aestivum (Amaryllidaceae) were investigated by capillary GC-MS. Excellent peak resolution for the alkaloids was exhibited and isomers of galanthamine and N-formylnorgalanthamine were well separated. Fourteen alkaloids of galanthamine, lycorine and crinane types were identified, 11 in the intact plants and eight in the in vitro cultures.
Abstract: A GC/MS analysis of alkaloids from leaves, bulbs and roots of Pancratium maritimum was performed. From the identified 16 alkaloids, 5 alkaloids were reported for the first time for this plant. Several compounds with pharmacological activity were found. Haemanthamine was main alkaloid in the leaves and bulbs whereas galanthane was found to be main alkaloid in roots.
Abstract: Alkaloid pattern of individuals from 16 Bulgarian Galanthus elwesii populations was investigated by GC/MS and TLC. Twenty-one Amaryllidaceae alkaloids were detected and 14 of them were identified. Crinane type alkaloids, haemanthamine or crinine, dominated alkaloid metabolism in most of the populations. With exception of one population, where the separate individuals showed variable alkaloid profiles (dominated by crinine or haemanthamine) the individuals of the rest of populations have identical and characteristic alkaloid profiles. Some populations showed remarkable differences in respect to their alkaloid pattern-type of biosynthesis, main alkaloids and number of alkaloids. Populations dominated by galanthamine type alkaloids were found as well. These data demonstrate that like the morphological features, the alkaloid metabolism of G. elwesii is also variable.
Abstract: A rapid and convenient method for sample preparation and simultaneous densitometric quantification of hyoscyamine (1) and scopolamine (2) has been developed. The alkaloid profiles of plant samples, as well as the content of 1 and 2, determined by TLC-densitometry corresponded well with those obtained using a GC method. The proposed method is simple and sensitive and can be used for the routine assay of 1 and 2, and for screening for alkaloid-rich and biochemically-interesting transformed root cultures and intact solanaceous plants.
Abstract: The alkaloid spectra of Datura innoxia plants grown in Egypt and Bulgaria were investigated by GC-MS. Thirty-eight alkaloids were detected in the roots, leaves and fruits of the plants. Five new alkaloids for D. innoxia are reported. Alkaloid spectra of Egyptian and Bulgarian plants differ significantly in respect to their alkaloid composition and main alkaloids accumulated in the plant organs.
Abstract: Thirty-six alkaloids were identified in the organs of Datura ceratocaula by GC/MS. Thirty-three of them have not been previously reported for the species. Furthermore, a new tropane ester was tentatively identified as 3-(3'-formyloxytropoyloxy)tropane on basis of its mass spectral fragmentation. Hyoscyamine was the main alkaloid in the plant organs.
Abstract: Sixteen populations of Galanthus elwesii Hook.fil and five populations of Galanthus nivalis L. growing in Bulgaria were investigated for the presence of galanthamine by TLC and GC-MS. Between 3 and 11 alkaloids were detected by TLC in the total alkaloid fractions. Galanthamine was found in 2 populations of G. elwesii.
Abstract: Hairy root cultures were obtained from diploid and induced tetraploid plants of Datura stramonium and analyzed by gas chromatography/mass spectrometry. Twenty alkaloids (19 for diploid and 9 for tetraploid hairy root cultures) were identified. A new tropane ester 3-tigloyloxy-6-propionyloxy-7-hydroxytropane was identified on the basis of mass spectral data. Hyoscyamine was the main alkaloid in both diploid and tetraploid cultures. In contrast to diploid hairy roots, the percentage contributions of the alkaloids, with exceptions for hyoscyamine and apoatropine, were higher in the total alkaloid mixture of tetraploid hairy roots.
Abstract: Alkaloids, GS-MS, Datura stramonium The alkaloid spectrum in roots, leaves and seeds of Datura stramonium L. was investigated by GC-MS. Twenty-nine tropane alkaloids are detected. Twelve of them are new constituents for the species and the two tropane esters 3-(3'-acetoxytropoyloxy)tropane (21) and 3-(2'-hydroxytropoyloxy)tropane (26) are described for the first time.
