The Anti-Cholinesterase Potential of Fifteen Different Species of Narcissus L. (Amaryllidaceae) Collected in Spain.

Narcissus L. is a renowned plant genus with a notable center of diversity and is primarily located in the Mediterranean region. These plants are widely recognized for their ornamental value, owing to the beauty of their flowers; nonetheless, they also hold pharmacological importance. In Europe, pharmaceutical companies usually use the bulbs of Narcissus pseudonarcissus cv. Carlton to extract galanthamine, which is one of the few medications approved by the FDA for the palliative treatment of mild-to-moderate symptoms of Alzheimer's disease. The purpose of this study was to evaluate the potential of these plants in Alzheimer's disease. The alkaloid extract from the leaves of different species of Narcissus was obtained by an acid-base extraction work-up -procedure. The biological potential of the samples was carried out by evaluating their ability to inhibit the enzymes acetyl- and butyrylcholinesterase (AChE and BuChE, respectively). The species N. jacetanus exhibited the best inhibition values against AChE, with IC50 values of 0.75 ± 0.03 µg·mL-1, while N. jonquilla was the most active against BuChE, with IC50 values of 11.72 ± 1.15 µg·mL-1.

Chemical and Biological Aspects of Different Species of the Genus Clinanthus Herb. (Amaryllidaceae) from South America.

The genus Clinanthus Herb. is found in the Andes Region (South America), mainly in Peru, Ecuador, and Bolivia. These plants belong to the Amaryllidaceae family, specifically the Amaryllidoideae subfamily, which presents an exclusive group of alkaloids known as Amaryllidaceae alkaloids that show important structural diversity and pharmacological properties. It is possible to find some publications in the literature regarding the botanical aspects of Clinanthus species, although there is little information available about their chemical and biological activities. The aim of this work was to obtain the alkaloid profile and the anti-cholinesterase activity of four different samples of Clinanthus collected in South America: Clinanthus sp., Clinanthus incarnatus, and Clinanthus variegatus. The alkaloid extract of each sample was analyzed by gas chromatography coupled with mass spectrometry (GC-MS), and their potential against the enzymes acetyl- and butyrylcholinesterase were evaluated. Thirteen alkaloids have been identified among these species, while six unidentified structures have also been detected in these plants. The alkaloid extract of the C. variegatus samples showed the highest structural diversity as well as the best activity against AChE, which was likely due to the presence of the alkaloid sanguinine. The results suggest this genus as a possible interesting new source of Amaryllidaceae alkaloids, which could contribute to the development of new medicines.

Gas chromatography-mass spectrometry of some homolycorine-type Amaryllidaceae alkaloids.

RATIONALE: Gas chromatography-mass spectrometry (GC-MS) is the most frequently applied technique for analyzing Amaryllidaceae alkaloids in plant extracts. Having these compounds, known for their potent bioactivities, is a distinctive chemotaxonomic feature of the Amaryllidoideae subfamily (Amaryllidaceae). The Amaryllidaceae alkaloids of homolycorine type with a C3-C4 double bond generally show molecular and diagnostic ions at the high-mass region with low intensity in the EIMS mode, leading to problematic identification in complex plant extracts. METHODS: Eleven standard homolycorine-type alkaloids (isolated and identified by 1D and 2D nuclear magnetic resonance) were subjected to separation with GC and studied with electron impact mass spectrometry (EIMS) including single quadrupole (GC-EIMS), tandem (GC-EIMS/MS), and high-resolution (GC-HR-EIMS) detectors, as well as with chemical ionization mass spectrometry (GC-CIMS). Alkaloid fractions from two Hippeastrum species and Clivia miniata were subjected to GC-EIMS and GC-CIMS for alkaloid identification. RESULTS: GC-EIMS in combination with GC-CIMS provided significant structural information of homolycorine-type alkaloids with C3-C4 double bond, facilitating their unambiguous identification. Based on the obtained typical fragmentation, other 11 homolycorine-type compounds were identified in extracts from two Hippeastrum species by parallel GC-EIMS, GC-CIMS, and liquid chromatography-electrospray ionization time-of-flight mass spectrometry and in extracts from C. miniata by GC-EIMS. CONCLUSIONS: GC-MS can be successfully applied for the identification of new and known homolycorine-type alkaloids, among others within the Amaryllidoideae subfamily, as well as for chemotaxonomical and chemoecological studies.

Alkaloid Profile in Wild Autumn-Flowering Daffodils and Their Acetylcholinesterase Inhibitory Activity.

Amaryllidaceae alkaloids are secondary metabolites with interesting medicinal properties. Almost every Narcissus species can synthesize them and constitute an excellent source for their isolation and study. Several Amaryllidaceae alkaloids have shown acetylcholinesterase inhibitory activities and are a promising tool for treating cholinergic disorders such as Alzheimer's disease (AD). Indeed, three of the four palliative treatments approved for AD are acetylcholinesterase (AChE) inhibitors and one of them, galanthamine, is an Amaryllidaceae alkaloid itself. This molecule is currently isolated from natural sources. However, its production is insufficient to supply the increasing demand for the active principle. Our main aim is to discover tools to improve galanthamine production and to prospect for potential new and more efficient drugs for AD treatment. Furthermore, we seek to broaden the knowledge of plants of the genus Narcissus from a chemotaxonomic perspective. Hence, in this study, we evaluate the alkaloid content through GC-MS and the AChE inhibitory activity of ten autumn-flowering Narcissus, which have been less studied than their spring-flowering counterparts. A total of thirty Amaryllidaceae alkaloids have been found, twenty-eight properly identified. Two Narcissus contained galanthamine, and seven were able to inhibit AChE.

Chemical Survey of Three Species of the Genus Rauhia Traub (Amaryllidaceae).

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 (IC50) values of 0.17 ± 0.02 µg·mL-1 in comparison with the IC50 values of 0.53 ± 0.12 µg·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.

GC-MS of some lycorine-type Amaryllidaceae alkaloids.

The search for novel bioactive compounds and the identification of known ones in the plant kingdom are a challenge for the scientists working in different fields of plant science. In the recent years, mass spectrometry is the most frequently applied method for analysis of complex mixtures of plant metabolites. Twenty-two alkaloids of different lycorine skeleton subtypes (with a Δ3,4 double bond, with a Δ4,11 double bond, with saturated rings C and D, and with aromatic ring C) were subjected to separation with gas chromatography and studied with electron impact mass spectrometry including single quadropole (GC-EIMS), tandem mass (GC-EIMS/MS) and high resolution mass (EI-HRMS) detectors in order to determine their fragmentation pattern. The compounds showed excellent separation and specific MS fragmentation allowing structural determination. The GC-MS can be successfully applied for searching new and identification of known bioactive molecules, chemotaxonomical and chemoecological studies, among others, within the Amaryllidoideae subfamily.

Chemodiversity, chemotaxonomy and chemoecology of Amaryllidaceae alkaloids.

The Amaryllidaceae alkaloids are a distinctive chemotaxonomic feature of the subfamily Amaryllidoideae of the family Amaryllidaceae, which consists of 59 genera and >800 species distributed primarily in tropical and subtropical areas. Since the first isolation, ca. 140 ago, >600 structurally diverse Amaryllidaceae alkaloids have been reported from ca. 350 species (44% of all species in the subfamily). A few have been found in other plant families, but the majority are unique to the Amaryllidoideae. These alkaloids have attracted considerable research interest due to their wide range of biological and pharmacological activities, which have been extensively reviewed. In this chapter we provide a review of the 636 structures of isolated or tentatively identified alkaloids from plants of the Amaryllidoideae and their classification into 42 skeleton types, as well as a discussion on their distribution, and chemotaxonomical and chemoecological aspects.

N-oxide alkaloids from Crinum amabile (Amaryllidaceae).

Natural products play an important role in the development of new drugs. In this context, the Amaryllidaceae alkaloids have attracted considerable attention in view of their unique structural features and various biological activities. In this study, twenty-three alkaloids were identified from Crinum amabile by GC-MS and two new structures (augustine N-oxide and buphanisine N-oxide) were structurally elucidated by NMR. Anti-parasitic and cholinesterase (AChE and BuChE) inhibitory activities of six alkaloids isolated from this species, including the two new compounds, are described herein. None of the alkaloids isolated from C. amabile gave better results than the reference drugs, so it was possible to conclude that the N-oxide group does not increase their therapeutic potential.

Analysis of Polyamines Conjugated with Hydroxycinnamoyl Acids by High-Performance Liquid Chromatography Coupled to Electrospray Ionization Tandem Mass Spectrometry.

Polyamines conjugated with hydroxycinnamic acids are phenolic compounds, which are widespread in the plant kingdom playing important roles in development and defence responses. This chapter describes the methodology employed to analyze these phenolamides in plant material by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-MS-MS). These compounds are not always in sufficient concentration in plant tissues for analysis by more conventional methods such as UV detection of HPLC. Owing to their particular molecular structure, they cannot be analyzed as free polyamines. Thus, described herein is an extraction method for hydroxycinnamic acid amides in plant tissues such as leaves, and their analysis by LC-MS-MS, including identification and quantification protocols.

Hippeastrum reticulatum (Amaryllidaceae): Alkaloid Profiling, Biological Activities and Molecular Docking.

The Amaryllidaceae family has proven to be a rich source of active compounds, which are characterized by unique skeleton arrangements and a broad spectrum of biological activities. The aim of this work was to perform the first detailed study of the alkaloid constituents of Hippeastrum reticulatum (Amaryllidaceae) and to determine the anti-parasitological and cholinesterase (AChE and BuChE) inhibitory activities of the epimers (6α-hydroxymaritidine and 6ß-hydroxymaritidine). Twelve alkaloids were identified in H. reticulatum: eight known alkaloids by GC-MS and four unknown (6α-hydroxymaritidine, 6ß-hydroxymaritidine, reticulinine and isoreticulinine) by NMR. The epimer mixture (6α-hydroxymaritidine and 6ß-hydroxymaritidine) showed low activity against all protozoan parasites tested and weak AChE-inhibitory activity. Finally, a molecular docking analysis of AChE and BuChE proteins showed that isoreticulinine may be classified as a potential inhibitory molecule since it can be stabilized in the active site through hydrogen bonds, π-π stacking and hydrophobic interactions.

Alkaloid Constituents of the Amaryllidaceae Plant Amaryllis belladonna L.

The plant family Amaryllidaceae is well-known for its unique alkaloid constituents, which exhibit a wide range of biological activities. Its representative, Amaryllis belladonna, has a geographical distribution covering mainly southern Africa, where it has significant usage in the traditional medicine of the native people. In this study, A. belladonna samples collected in Brazil were examined for alkaloid content. Alkaloid profiles of A. belladonna bulbs were generated by a combination of chromatographic, spectroscopic and spectrometric methods, including GC-MS and 2D NMR. In vitro screening against four different parasitic protozoa (Trypanosoma cruzi, T. brucei rhodesiense, Leishmania donovani and Plasmodium falciparum) was carried out using the A. belladonna crude methanol extract, as well as three of its alkaloid isolates. Twenty-six different Amaryllidaceae alkaloids were identified in the A. belladonna bulb samples, and three of them were isolated. Evidence for their respective biosynthetic pathways was afforded via their mass-spectral fragmentation data. Improved data for 1-O-acetylcaranine was provided by 2D NMR experiments, together with new ¹H-NMR data for buphanamine. The crude extract and 3-O-acetylhamayne exhibited good antiprotozoal activity in vitro, although both with a high cytotoxic index.

Crinine-type alkaloids from Hippeastrum aulicum and H. calyptratum.

An ongoing search for alkaloids in the Amaryllidaceae species using GC-MS resulted in the identification of two crinine-type alkaloids, aulicine (1) and 3-O-methyl-epimacowine, (2) from the indigenous Brazilian species Hippeastrum aulicum and Hippeastrum calyptratum, respectively. In addition, two alkaloids, 11-oxohaemanthamine (3) and 7-methoxy-O-methyllycorenine (4) were both isolated from H. aulicum. Furthermore, we provide here complete NMR spectroscopic data for the homolycorine analogues nerinine (5) and albomaculine (6). The absolute stereochemistry of the 5,10b-ethano bridge in the crinine variants was determined by circular dichroism and X-ray crystallographic analysis, thus presenting the first direct evidence for the presence of crinine-type alkaloids in the genus Hippeastrum.

Alkaloids of the South African Amaryllidaceae: a review.

The plant family Amaryllidaceae is known for its horticultural and ornamental appeal as well as its medicinal value. In relation to these characteristics, trade in Amaryllid flower varieties (especially daffodils) is a multi-million dollar revenue generator for the floriculture industry. Of greater significance are the medicinal attributes of the family, which has already spawned the Alzheimer's prescription drug galanthamine, a potent and selective inhibitor of the enzyme acetylcholinesterase, of significance in the progression of neurodegeneration associated with motor neuron diseases, with annual global sales of around $150 million. Furthermore, it is anticipated that an anticancer drug target related to the Amaryllidaceae alkaloid pancratistatin, presently under advanced clinical evaluation, will enter commercial circulation within the next decade. Members of the Amaryllidaceae are distributed through both tropical and subtropical regions of the globe, but are of prominence within three distinct geographical locations, including Andean South America, the Mediterranean basin, and southern Africa. The southern African zone is known to harbor at least a third of the worldwide complement of around 1000 species, many of which are widely utilized in the traditional medicinal practices of the indigenous people of the region. Given its therapeutic and economic value, its natural abundance in the southern African region, coupled to its widespread usage in ethnic medicine, the family Amaryllidaceae provides a diverse and accessible platform for phytochemical based drug discovery. A consolidation of its traditional usage as well as its chemical and pharmacological profiles will thus guide efforts aimed at maximizing this potential. In undertaking this survey of the Amaryllidaceae of southern African, we aimed to achieve these goals.

Wild daffodils of the section Ganymedes from the Iberian Peninsula as a source of mesembrane alkaloids.

The aim of this work was to perform a detailed study of the alkaloid content of Narcissus triandrus, as well as a complete analysis of the alkaloid profile of 18 wild populations, comprising all the taxa of the section Ganymedes. Through the application of a combination of spectroscopic and chromatographic methods, the isolation and structural elucidation of 3 compounds are reported for the first time from a natural source (2-oxomesembrenone, 7,7a-dehydromesembrenone and 2-oxoepimesembranol), together with the identification of 5 major common mesembrane alkaloids. Additionally, the GC-MS analysis of the alkaloid profile demonstrated the regular presence of mesembranes in all the studied plants, showing mesembrenone as the predominant compound without any typical Amaryllidaceae alkaloid being detected.

GC-MS of amaryllidaceous galanthamine-type alkaloids.

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.

Alkaloids from Narcissus serotinus.

Narcissus serotinus belongs to the Amaryllidaceae family, a group well known for an exclusive variety of alkaloids with interesting biological activities. This study was aimed at identifying the alkaloid constituents of N. serotinus collected in the Spanish region of Valencia, using a combination of chromatographic, spectroscopic, and spectrometric methods, including GC-MS and 2D NMR techniques. GC-MS analysis allowed for the direct identification of five known compounds. In addition, the isolation and structure elucidation of six new Amaryllidaceae alkaloids are described.

Bioactive alkaloid extracts from Narcissus broussonetii: mass spectral studies.

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 1.77 µg/ml.

Analysis of phenolic compounds by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry in senescent and water-stressed tobacco.

Evaluation of a significant part of the phenylpropanoid pathway metabolites is facilitated by the fast high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS) analytical method. The technology described was applied in tobacco plants (Nicotiana tabacum L. cv. Wisconsin) to identify 20 phenolic compounds and to detect differences in phenylpropanoid profiles in two types of experiments. In the first one, senescent and non-senescent parts of flowering plants were compared, while in the second, watered plants were compared with water-stressed young plants. The 20 identified phenolic compounds were: seven hydroxycinnamoylquinic acids, seven hydroxycinnamic acid glucosides, one salicylic acid glucoside, two conjugated flavonols with disaccharides, and three hydroxycinnamic acid amides (HCAA) of putrescine. In general, the levels of phenylpropanoid compounds increased under water stress or senescent conditions, with the exception of HCAA, which decreased in senescent samples, and 4-O-p-coumaroylquinic acid and trihydroxycinamic acid-O-glucoside, which did not change in both experiments. The main product in all the samples was 5-O-caffeoylquinic acid (neochlorogenic acid). Another compound, kaempferol-7-O-neohesperidoside, was tentatively identified for the first time in tobacco plants. This method, which can be applied in other plant species, allows a simple and efficient comparative study of metabolite profile variations (qualitative and quantitative) in response to different physiological and/or environmental plant situations.

Acetylcholinesterase-inhibiting alkaloids from Zephyranthes concolor.

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 × 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.

Alkaloids from Hippeastrum papilio.

Galanthamine, an acetylcholinesterase inhibitor marketed as a hydrobromide salt (Razadyne®, Reminyl®) 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 11ß-hydroxygalanthamine, an epimer of the previously isolated alkaloid habranthine, which was identified using NMR techniques. It has been shown that 11ß-hydroxygalanthamine has an important in vitro acetylcholinesterase inhibitory activity. Additionally, Hippeastrum papilio yielded substantial quantities of galanthamine.