Acetylcholinesterase (AChE) is one of the classical targets in the treatment of Alzheimer's disease (AD). Inhibition of AChE slows down the hydrolysis of acetycholine and increases choline levels, improving the cognitive function. The achieved success of plant-based natural drugs acting as AChE inhibitors, such as galantamine (GAL) from Galanthus genus and huperzine A from Huperzia serrate (approved drug in China), in the treatment of AD, and the fact that natural compounds (NCs) are considered as safer and less toxic compared to synthetic drugs, led us to screen the available NCs (almost 150,000) in the ZINC12 database for AChE inhibitory activity. The compounds were screened virtually by molecular docking, filtered for suitable ADME properties, and 32 ligands from 23 structural groups were selected. The stability of the complexes was estimated via 1 micros molecular dynamics simulation. Ten compounds formed stable complexes with the enzyme and had a vendor and a reasonable price per mg. They were tested for AChE inhibitory and antioxidant activity. Five compounds showed weak AChE inhibition and three of them exhibited high antioxidant activity.
Plant biodiversity is an important source of compounds with medicinal properties. The alkaloid galanthamine, first isolated from Galanthus woronowii (Amaryllidaceae), is approved by the FDA for the palliative treatment of mild to moderate Alzheimer's disease due to its acetylcholinesterase (AChE) inhibitory activity. Obtaining this active pharmaceutical ingredient, still sourced on an industrial scale from the Amaryllidaceae species, is a challenge for pharmaceutical companies due to its low natural yield and the high cost of its synthesis. The aim of this work was to determine the alkaloid profile of three different Rauhia (Amaryllidaceae) species collected in Peru, and to assess the potential application of their extracts for the treatment of Alzheimer's disease. The alkaloids were identified by gas chromatography coupled to mass spectrometry (GC-MS), and the AChE inhibitory activity of the extracts was analyzed. Thirty compounds were quantified from the Rauhia species, the R. multiflora extract being the most interesting due to its high diversity of galanthamine-type structures. The R. multiflora extract was also the most active against AChE, with the half maximal inhibitory concentration (IC(50)) values of 0.17 +/- 0.02 microg.mL(-1) in comparison with the IC(50) values of 0.53 +/- 0.12 microg.mL(-1) for galanthamine, used as a reference. Computational experiments were carried out on the activity of the galanthamine-type alkaloids identified in R. multiflora toward five different human AChE structures. The simulation of the molecules 3-O-acetylgalanthamine, 3-O-acetylsanguinine, narwedine, and lycoraminone on the 4EY6 crystal structure theoretically showed a higher inhibition of hAChE and different interactions with the active site compared to galanthamine. In conclusion, the results of this first alkaloid profiling of the Rauhia species indicate that R. multiflora is an important natural source of galanthamine-type structures and could be used as a model for the development of biotechnological tools necessary to advance the sustainable production of galanthamine.
AIMS: Amaryllidaceae alkaloids exhibit a wide range of physiological effects, of which the acetylcholinesterase (AChE) inhibitory activity is the most relevant. However, scientific evidence related to their neuroprotective effectiveness against glutamate-induced toxicity has been lacking. Thus, the purpose of this study was to conduct a comparative study of the neuroprotective activity and the AChE inhibitory activity of species of Amaryllidaceae. MAIN METHODS: The neuroprotective activity against glutamate-induced toxicity was measured in rat cortical neurons and the Ellman method was employed for the quantification of acetylcholinesterase inhibitory activity of alkaloidal extracts of five species of Amaryllidaceae (Crinum jagus, Crinum bulbispermum, Hippeastrum barbatum, Hippeastrum puniceum and Zephyranthes carinata). The alkaloid Amaryllidaceae patterns based on GC/MS analyses were also investigated. KEY FINDINGS: The results showed that the alkaloidal extract from C. jagus presented a high neuroprotective activity in both pre- and post-treatments against a glutamate excitotoxic stimulus. Furthermore, the alkaloid extracts from C. jagus and Z. carinata revealed an inhibitory activity of AChE from the electric eel with IC50 values of 18.28+/-0.29 and 17.96+/-1.22mug/mL, respectively. In addition, 46 alkaloids were detected by GC/MS, and 20 of them were identified based on their mass spectra and retention index. The results suggest that the neuroprotective effects might be associated with lycorine and crinine-type alkaloids, whereas the acetylcholinesterase enzyme inhibitory activity could be related to galanthamine and lycorine-type alkaloids, although not based on synergistic processes. SIGNIFICANCE: In summary, Amaryllidaceae species are sources of alkaloids with potential use for Alzheimer's disease.
A GC-MS analysis of alkaloids in the aerial parts and bulbs of Galanthus xvalentinei nothosubsp. subplicatus was performed for the first time. Totally, twenty-six alkaloids were identified, of which tazettine and galanthindole were the major ones. Acetylcholinesterase inhibitory activity of the alkaloidal extracts was determined using modified in vitro Ellman's method. Significant anticholinesterase activity was observed in the tested samples (bulbs: IC50 = 21.3 microg/mL, aerial parts: IC50 = 16.3 microg/mL).
GC-MS (gas chromatography-mass spectrometry) analyses of alkaloids in the aerial parts and bulbs of Galanthus rizehensis Stern (Amaryllidaceae), collected during two different vegetation periods, was performed. Twenty three alkaloids were identified in four different alkaloid extracts. Acetylcholinesterase (AChE) inhibitory activities of the alkaloid extracts were tested. Both the highest alkaloid diversity and the most potent inhibitory activity (IC50 12.94 microg/ml) were obtained in extracts from the bulbs of G. rizehensis collected during the fruiting period.
Galanthamine-type alkaloids produced by plants of the Amaryllidaceae family are potent acetylcholinesterase inhibitors. One of them, galanthamine, has been marketed as a hydrobromide salt for the treatment of Alzheimer's disease. In the present work, gas chromatography with electron impact mass spectrometry (GC-EIMS) fragmentation of 12 reference compounds isolated from various amaryllidaceous plants and identified by spectroscopic methods (1D and 2D nuclear magnetic resonance, circular dichroism, high-resolution MS (HRMS) and EIMS) was studied by tandem mass spectrometry (GC-MS/MS) and accurate mass measurements (GC-HRMS). The studied compounds showed good peak shape and efficient GC separation with a GC-MS fragmentation pattern similar to that obtained by direct insertion probe. With the exception of galanthamine-N-oxide and N-formylnorgalanthamine, the galanthamine-type compounds showed abundant [M](+.) and [M-H](+) ions. A typical fragmentation pattern was also observed, depending on the substituents of the skeleton. Based on the fragmentation pathways of reference compounds, three other galanthamine-type alkaloids, including 3-O-(2'-butenoyl)sanguinine, which possesses a previously unelucidated structure, were identified in Leucojum aestivum ssp. pulchelum, a species endemic to the Balearic islands. GC-MS can be successfully applied to Amaryllidaceae plant samples in the routine screening for potentially new or known bioactive molecules, chemotaxonomy, biodiversity and identification of impurities in pharmaceutical substances.
The Amaryllidaceae family is well known for its pharmacologically active alkaloids An important approach to treat Alzheimer&'s disease involves the inhibition of the enzyme acetylcholinesterase AChE Galanthamine an Amaryllidaceae alkaloid is an effective selective reversible and competitive AchE inhibitor This work was aimed at studying the alkaloid composition of four wild Argentinian Amarillydaceae species for the first time as well as analyzing their inhibitory activity on acetylcholinesterase Alkaloid content was characterized by means of GC-MS analysis Chloroform basic extracts from Habranthus jamesonii Phycella herbertiana Rhodophiala mendocina and Zephyranthes filifolia collected in the Argentinian Andean region all contained galanthamine and showed a strong AChE inhibitory activity IC(50 between 1.2 and 2 181;g/mL To our knowledge no previous reports on alkaloid profiles and AChEIs activity of wild Argentinian Amarillydaceae species have been publisihed The demand for renewable sources of industrial products like galanthamine and the need to protect plant biodiversity creates an opportunity for Argentinian farmers to produce such crops.
Plants of the Amaryllidaceae family are a well-known source of tetrahydroisoquinoline alkaloids with a wide range of biological activities including antiviral antitumoral antiparasitic psychopharmacological and acetylcholinesterase inhibitory among others Recent advances in the use of GC or LC coupled to MS have allowed a chemically guided isolation of uncommon and bioactive alkaloids In the present work analytical methods were applied to study the alkaloid profile of Narcissus broussonetii a plant endemic to North Africa Using the GC-MS technique and an in-home mass fragmentation database twenty-three alkaloids were identified including the very rare dinitrogenous alkaloids obliquine plicamine and secoplicamine Applying LC-ESI-LTQ-Orbitrap-MS fragmentation profiles were found to be similar for obliquine and plicamine but different for secoplicamine Pretazettine a potent cytotoxic alkaloid was also isolated from N broussonetii although its identification by GC-MS was only possible after a BSTFA-derivatization The silylated crude methanolic extract only showed the presence of pretazettine-TMS confirming that tazettine was formed after the alkaloid extraction The same observation was made in Narcissus cultivars in which tazettine had been detected as the major alkaloid As part of an ongoing project on MS of Amaryllidaceae alkaloids the silylated tazettine and pretazettine were studied by GC-MS/MS and found to differ in their fragmentation routes Finally the EtOAc extract of N broussonetii showed notable in vitro activity against Trypanosoma cruzi with an IC(50 value of 1.77mug/ml.
Title: Development and validation of a GC-MS method for rapid determination of galanthamine in Leucojum aestivum and Narcissus ssp.: a metabolomic approach Berkov S, Bastida J, Viladomat F, Codina C Ref: Talanta, 83:1455, 2011 : PubMed
Galanthamine, an acetylcholinesterase inhibitor marketed as a hydrobromide salt for the treatment of Alzheimer's disease, is obtained from some Amaryllidaceae plants. A new method was developed and validated for its quantification by GC-MS in different plant sources: bulbs and leaves from Narcissus confusus; bulbs from N. pseudonarcissus cv. Carlton; and leaves and in vitro cultures from L. aestivum. Samples (50 mg) were extracted with methanol (1 mL) for 2 h, then aliquots of the extracts were silylated and analyzed by GC-MS. The calibration line was linear over a range of 15-800 mug galanthamine/sample, ensuring an analysis of samples with a content of 0.03-1.54% analyte referred to dry weight. The recovery was generally more than 95%. Good inter- and intra assay precision was observed (RSD<3%). Principal component analysis of GC-MS chromatograms allowed discrimination of the plant raw material with respect to species, organs and geographical regions. The analytical method developed in this study proved to be simple, sensitive and far more informative than the routine analytical methods (GC, HPLC, CE and NMR), so it may be useful for quality control of plant raw materials in the pharmaceutical industry.
The bulbs and aerial parts of Zephyranthes concolor (Lindl.) Benth. & Hook. f. (Amaryllidaceae), an endemic species to Mexico, were found to contain the alkaloids chlidanthine, galanthamine, galanthamine N-oxide, lycorine, galwesine, and epinorgalanthamine. Since currently only partial and low resolution (1)H-NMR data for chlidanthine acetate are available, and none for chlidanthine, its 1D and 2D high resolution (1)H- and (13)C-NMR spectra were recorded. Unambiguous assignations were achieved with HMBC, and HSQC experiments, and its structure was corroborated by X-ray diffraction. Minimum energy conformation for structures of chlidanthine, and its positional isomer galanthamine, were calculated by molecular modelling. Galanthamine is a well known acetylcholinesterase inhibitor; therefore, the isolated alkaloids were tested for this activity. Chlidanthine and galanthamine N-oxide inhibited electric eel acetylcholinesterase (2.4 and 2.6 x 10(-5) M, respectively), indicating they are about five times less potent than galanthamine, while galwesine was inactive at 10(-3) M. Inhibitory activity of HIV-1 replication, and cytotoxicity of the isolated alkaloids were evaluated in human MT-4 cells; however, the alkaloids showed poor activity as compared with standard anti-HIV drugs, but most of them were not cytotoxic.
Galanthamine, an acetylcholinesterase inhibitor marketed as a hydrobromide salt (Razadyne(R), Reminyl(R)) for the treatment of Alzheimer's disease (AD), is obtained from Amaryllidaceae plants, especially those belonging to the genera Leucojum, Narcissus, Lycoris and Ungernia. The growing demand for galanthamine has prompted searches for new sources of this compound, as well as other bioactive alkaloids for the treatment of AD. In this paper we report the isolation of the new alkaloid 11beta-hydroxygalanthamine, an epimer of the previously isolated alkaloid habranthine, which was identified using NMR techniques. It has been shown that 11beta-hydroxygalanthamine has an important in vitro acetylcholinesterase inhibitory activity. Additionally, Hippeastrum papilio yielded substantial quantities of galanthamine.
Title: Analysis of galanthamine-type alkaloids by capillary gas chromatography-mass spectrometry in plants Berkov S, Bastida J, Viladomat F, Codina C Ref: Phytochem Anal, 19:285, 2008 : PubMed
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.
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).
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.
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.
