Rapid TLC/GC-MS identification of acetylcholinesterase inhibitors in alkaloid extracts

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.     

Alkaloid variability in Leucojum aestivum from wild populations

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.     

Choline esterase inhibitory properties of alkaloids from two Nigerian Crinum species

The bulbs of Crinum jagus and Crinum glaucum are used in traditional medicine in southern Nigeria for memory loss and other mental symptoms associated with ageing. Alkaloidal extracts of bulbs from each species showed inhibition of acetylcholinesterase, an activity exploited therapeutically to raise the depressed levels of acetylcholine in the brain associated with Alzheimer's disease. Using the in situ bioautographic test method for enzyme inhibition, a number of alkaloids were isolated and their activity quantified using the Ellman spectrophotometric test. The most active alkaloids isolated were hamayne (IC50 250 microM) and lycorine (IC50 450 microM) whilst other alkaloids were comparatively inactive with haemanthamane giving 3% inhibition and crinamine giving 4.4% inhibition at 50 mg ml(-1) (174 microM). These contrast with the positive control physostigmine which gave IC50 of 0.25 microM. Cholinesterase activity appears to be associated with the presence of two free hydroxy groups in this structural type of Amaryllidaceae alkaloid.     

Effects of sucrose and plant growth regulators on acetylcholinesterase inhibitory activity of alkaloids accumulated in shoot cultures of Amaryllidaceae

The influence of sucrose (30, 60, 90 and 120 g/L), activated charcoal (5 and 10 g/L), and various levels of several plant growth regulators (6-benzyladenine, naphthalene-1-acetic acid, 2,4-dichlorophenoxyacetic acid, and picloram) on organogenesis (bulb and root development) and the accumulation of alkaloid and galanthamine in shoot cultures of three Amaryllidaceae species (Narcissus pseudonarcissus, Galanthus elwesii, and Leucojum aestivum) was investigated in a full-factorial experiment. Alkaloid extracts were analyzed by gas chromatography–mass spectrometry, leading to the quantification of galanthamine and to the identification of other alkaloids. The different extracts were then subjected to an Ellman test to evaluate the inhibitory activity of acetylcholinesterase. The highest contents of galanthamine [0.02–0.1% dry weight (DW) depending on the plant species] were always accompanied with a high level of acetylcholinesterase inhibition (>30%). However, some samples containing low amounts of galanthamine (0.005% DW) showed high inhibitory activities (40–80%). These findings demonstrate the presence of Amaryllidaceae alkaloids that have not yet been identified as having anti-acetylcholinesterase activity.     

Selective cytochrome P450 3A4 inhibitory activity of Amaryllidaceae alkaloids

A library of natural and semi-synthetic Amaryllidaceae alkaloids was screened for cytochrome P450 3A4 (CYP3A4) inhibitory activity. Of the crinane, lycorane and galanthamine representatives examined two semi-synthetic silylated lycorane analogues, accessed via a chemoselective silylation strategy from lycorine, and the natural compound narciclasine exhibited low micromolar activities. Important pharmacological features uncovered include the lack of CYP3A4 inhibitory activity seen for galanthamine and the selective activity that is seen with narciclasine over pancratistatin.     

Alkaloids biosynthesis by <Emphasis Type="Italic">Pancratium maritimum</Emphasis> L. shoots in liquid culture

The process of alkaloid biosynthesis by Pancratium maritimum shoot culture, cultivated under submerged conditions, was investigated. Twenty-two compounds of different structural types of the Amaryllidaceae alkaloids (tyramine, narciclasine, galanthamine, haemanthamine, lycorine, pancracine, tazettine and homolycorine types) were detected in the studied samples from biomass and cultural liquid. Dominant compounds in the shoots were of tyramine, lycorine and haemanthamine types, whereas in the culture media were found mainly lycorine type compounds. Based on the multi-metabolic estimation of the alkaloid metabolism and physiological peculiarities, liquid cultures of P. maritimum shoots could be defined as prospective biological systems for producing bioactive molecules with acetylcholinesterase inhibitory and apoptotic activities.     

Galanthamine production by Leucojum aestivum L. shoot culture in a modified bubble column bioreactor with internal sections

Shoot culture of summer snowflake (Leucojum aestivum L.) was successfully cultivated in an advanced modified glass‐column bioreactor with internal sections for production of Amaryllidaceae alkaloids. The highest amounts of dry biomass (20.8 g/L) and galanthamine (1.7 mg/L) were achieved when shoots were cultured at 22°C and 18 L/(L·h) flow rate of inlet air. At these conditions, the L. aestivum shoot culture possessed mixotrophic‐type nutrition, synthesizing the highest amounts of chlorophyll (0.24 mg/g DW (dry weight) chlorophyll A and 0.13 mg/g DW chlorophyll B). The alkaloids extract of shoot biomass showed high acetylcholinesterase inhibitory activity (IC₅₀ = 4.6 mg). The gas chromatography–mass spectrometry (GC/MS) profiling of biosynthesized alkaloids revealed that galanthamine and related compounds were presented in higher extracellular proportions while lycorine and hemanthamine‐type compounds had higher intracellular proportions. The developed modified bubble‐column bioreactor with internal sections provided conditions ensuring the growth and galanthamine production by L. aestivum shoot culture.     

Preparation of secolycorines against acetylcholinesterase

5,6-Secolycorines possessing a 5,6-dihydrophenanthridine skeleton were facilely prepared from lycorine through chemical transformations, mainly including N-alkylation, Hofmann degradation type reaction, reductive cleavage of trichloroethylcarbonyl moiety, and hydrogenation. Several secolycorine derivatives showed potent inhibitory activity against acetylcholinesterase with the IC(50) value at micromolar range and are more potent than galanthamine.     

A rapid TLC bioautographic method for the detection of acetylcholinesterase and butyrylcholinesterase inhibitors in plants

A simple and rapid bioautographic enzyme assay on TLC plates has been developed for the screening of acetylcholinesterase and butyrylcholinesterase inhibition by plant extracts. Enzyme activity was detected by the conversion of naphthyl acetate into naphthol and the formation of the corresponding purple-coloured diazonium dye with Fast Blue B salt. Inhibitors of cholinesterases produced white spots on the dye-coloured background of the TLC plates. The alkaloids galanthamine and physostigmine, which are known inhibitors of acetylcholinesterase, were used to determine the sensitivity of the assay. Various plant extracts were tested using the bioassay.     

CGC-MS of alkaloids in Leucojum aestivum plants and their in vitro cultures

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.     

LED Light Quality Affect Growth,Alkaloids Contents,and Expressions of Amaryllidaceae Alkaloids Biosynthetic Pathway Genes in Lycoris longituba

Journal of Plant Growth Regulation - Lycoris longituba is an important ornamental and medicinal plant and contains various Amaryllidaceae alkaloids such as galanthamine (GAL), lycorine (LYC), and...     

Three-dimensional structure of a complex of galanthamine (Nivalin) with acetylcholinesterase from Torpedo californica: implications for the design of new anti-Alzheimer drugs

The 3D structure of a complex of the anti-Alzheimer drug galanthamine with Torpedo californica acetylcholinesterase is reported. Galanthamine, a tertiary alkaloid extracted from several species of Amarylidacae, is so far the only drug that shows a dual activity, being both an acetylcholinesterase inhibitor and an allosteric potentiator of the nicotinic response induced by acetylcholine and competitive agonists. The X-ray structure, at 2.5A resolution, shows an unexpected orientation of the ligand within the active site, as well as unusual protein-ligand interactions. The inhibitor binds at the base of the active site gorge, interacting with both the acyl-binding pocket and the principal quaternary ammonium-binding site. However, the tertiary amine group of galanthamine does not directly interact with Trp84. A docking study using the program AUTODOCK correctly predicts the orientation of galanthamine in the active site. The docked lowest-energy structure has a root mean square deviation of 0.5A with respect to the corresponding crystal structure of the complex. The observed binding mode explains the affinities of a series of structural analogs of galanthamine and provides a rational basis for structure-based drug design of synthetic derivatives with improved pharmacological properties. Proteins 2001;42:182-191.     

Using a phylogenetic approach to selection of target plants in drug discovery of acetylcholinesterase inhibiting alkaloids in Amaryllidaceae tribe Galantheae

We present phylogenetic analyses of 32 taxa of Amaryllidaceae tribe Galantheae, 6 taxa of other Eurasian genera of Amaryllidaceae and Phaedranassa dubia as outgroup in order to provide a phylogenetic framework for selection of candidate plants for lead discovery in relation to Alzheimer’s disease. We used DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS) and the plastid matK and trnL-F regions. Phylogenetic analyses using maximum parsimony and Bayesian inference strongly support a monophyletic tribe Galantheae in a narrow sense, including only Acis, Galanthus and Leucojum. Infrageneric relationships of Galanthus only partly support previous classifications. Alkaloid profiles and inhibition of acetylcholinesterase (AChE) were investigated for 18 taxa using gas chromatography–mass spectrometry (GC–MS) and an assay measuring inhibition of AChE activity. AChE inhibitory activity was found in all investigated clades and was correlated with alkaloid profiles of the plants. Lowest IC₅₀ values were expressed by extracts containing either galanthamine or lycorine type compounds. Evaluation of available chemistry and activity data in a phylogenetic framework could be used to select target species for further investigation.     

The geographic isolation of Leucojum aestivum populations leads to divergation of alkaloid biosynthesis

Leucojum aestivum, an industrial source of the acetylcholinesterase inhibitor galanthamine, shows a great chemodiversity in its alkaloid synthesis. Samples from various geographically distinct populations from Bulgaria and the Balearic Islands were studied by GC–MS. The alkaloid pattern of the plants of L. aestivum (subsp. pulchellum) from the Balearic Islands were dominated by crinine type compounds. Populations of homolycorine chemotype were distributed along the Danube river in the north part of Bulgaria, which is separated from the south part by the Balkan mountains. Populations with high accumulation of lycorine were found in East Bulgaria near the sea coast, while the south populations were dominated by galanthamine type synthesis. The average of the galanthamine content was found to vary from 0.003 to 0.08% (referred to dry weight) in the north, and up to 0.42% in the southern Bulgarian populations. Some individuals showed up to 0.65% galanthamine. The galanthamine content of the plants from the Balearic island was 0.1% of DW. The galanthamine percentage in the total alkaloid mixture ranged from 0.2 to 95% of the total alkaloids. Our study demonstrated that the geographic isolation of the populations of L. aestivum has led to divergation in the alkaloid biosynthesis and consequently to the occurrence of different chemotypes. This chemodiversity in both alkaloid patterns and galanthamine content provides an opportunity for further selection work toward a galanthamine-rich crop, on the one hand, and makes the species an excellent biological system for molecular studies leading to further improvement of the galanthamine production, which is a valuable alkaloid used in medicine for the treatment of Alzheimer's disease.     

Petrosamine, a potent anticholinesterase pyridoacridine alkaloid from a Thai marine sponge Petrosia n. sp

Two pyridoacridine alkaloids, including a known petrosamine and a new 2-bromoamphimedine were isolated from a Thai marine sponge Petrosia n. sp. The alkaloids were characterized on the basis of 1D and 2D NMR, MS, and IR spectroscopy. Only petrosamine showed strong acetylcholinesterase inhibitory activity approximately six times higher than that of the reference galanthamine. A computational docking study of petrosamine with the enzyme from the electric eel Torpedo californica (TcAChE) showed the major contribution to the petrosamine-TcAChE interaction to be arising from the quaternary ammonium group of petrosamine.     

Alkaloid patterns in Leucojum aestivum shoot culture cultivated at temporary immersion conditions

The alkaloid patterns in Leucojum aestivum L. shoot culture cultivated at temporary immersion conditions were investigated using gas chromatography-mass spectrometry. 18 alkaloids were identified, and galanthamine, hamayne and lycorine were dominant. The L. aestivum 80 shoot culture, cultivated at temporary immersion conditions, is a prospective biological matrix for obtaining wide range Amaryllidaceae alkaloids, showing valuable biological and pharmacological activities. The temperature of cultivation influenced enzyme activities, catalyzing phenol oxidative coupling of 4′-O-methylnorbelladine and formation of the different groups Amaryllidaceae alkaloids. Decreasing the temperature of cultivation of L. aestivum 80 shoot culture led to activation of para-ortho’ phenol oxidative coupling (formation of galanthamine type alkaloids) and inhibited ortho-para’ and para-para’ phenol oxidative coupling (formation of lycorine and haemanthamine types alkaloids).     

In vitro anticholinesterase activity of various alkaloids

In the current study, a number of alkaloids including retamine, cytisine, and sparteine (quinolizidine-type), yohimbine and vincamine (indole-type), scopolamine and atropine (tropane-type), colchicine (tropolone-type), allantoin (imidazolidine-type), trigonelline (pyridine-type) as well as octopamine, synephrine, and capsaicin (exocyclic amine-type) were tested in vitro for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at 1 mg/ml concentration by the Ellman method using an ELISA microplate reader. Among the alkaloids tested, only capsaicin exerted a remarkable inhibitory effect towards both AChE and BChE [(62.7 +/- 0.79)% and (75.3 +/- 0.98)%, respectively]. While the rest of the alkaloids did not show any significant inhibition against AChE, three of the alkaloids, namely retamine, sparteine, and yohimbine, exerted a noteworthy anti-BChE effect as compared to galanthamine, the reference drug.     

Temporary immersion systems for Amaryllidaceae alkaloids biosynthesis by Pancratium maritimum L. shoot culture

The alkaloid patterns of sea daffodil (Pancratium maritimum L.) shoot culture, cultivated in a temporary immersion cultivation system were investigated. The shoots accumulated maximal amounts of biomass (0.8 g dry biomass/L and Growth Index?=?1.6) at immersion frequency with 15 min flooding and 12 h stand-by periods. At this regime P. maritimum shoots achieved the highest degree of utilization of carbon source. Twenty-two alkaloids, belonging to narciclasine, galanthamine, haemanthamine, lycorine, montanine, tazettine, homolycorine and tyramine types were identified in intracellular and extracellular alkaloid extracts. The immersion frequency affected strongly the capacity of alkaloid biosynthesis in P. maritimum shoots and at the optimum conditions of cultivation, the total intracellular alkaloid content reached up to 3,469 μg/g dry biomass. The main biosynthesized alkaloids were haemanthamine (900.1 μg/g) and lycorine (799.9 μg/g). The obtained results proved that temporary immersion technology, as a cultivation approach, and P. maritimum shoots, as a biological system, are prospective for producing wide range bioactive alkaloids.     

Adventitious shoot regeneration from in vitro leaf explants of <Emphasis Type="Italic">Fraxinus nigra</Emphasis>

In order to establish an attractive method for the production of valuable medicinal alkaloids (galanthamine and lycorine), the plants of Leucojum aestivum and L. aestivum ‘Gravety Giant’ grown in bioreactor RITA® were subjected to various concentrations of methyl jasmonate (MeJA), salicylic acid (SA), 1-aminocyclopropane-1-carboxylic acid (ACC) and 2-chloroethylphosphonic acid (ethephon) at different times of culture. The application of MeJA showed a negative effect on L. aestivum and L. aestivum ‘Gravety Giant’ plant growth. We observed that the incubation of plants during 168 h with 100 µM of MeJA resulted above two times lower F.W. (fresh weight) increments compared with control. While SA showed an inhibitory effect only on the growth of L. aestivum cultures. ACC and ethephon had a positive effect on both types of culture. Treatment with 50 µM of MeJA during 168 h stimulated galanthamine and lycorine biosynthesis in L. aestivum and L. aestivum ‘Gravety Giant’ cultures. In addition, the accumulation of galanthamine was increased when 10 µM of ACC were added to both types of culture. 10 µM of ACC stimulated also lycorine biosynthesis by L. aestivum ‘Gravety Giant’. The addition of 10 µM of ethephon had a positive effect only on lycorine production in plants of L. aestivum. SA promoted galanthamine and lycorine biosynthesis in tested plants. Indeed the highest galanthamine (0.8 mg/g dry weight: D.W.) and lycorine (1.53 mg/g D.W.) concentrations were observed in L. aestivum ‘Gravety Giant’ plants treated with 5 µM of SA during 10 h.