Amaryllidaceae Plants Research Articles

My Favorite Enzyme: For love of lectins

I was a student of Fred Smith at the University of Minnesota during 1952–1959. Smith was an Englishman who came to the USA to work on the Manhattan Project to build the atomic bomb. He was an outstanding carbohydrate chemist who was mentored by Sir Norman Haworth, Nobel Laureate in Chemistry for the synthesis of vitamin C (ascorbic acid). Smith was at the University of Birmingham when he received the call to come to the United States to work on ‘‘the bomb’’ because of his expertise in fluorine chemistry, a necessary technique for the separation of the 238 isotope of uranium as the hexafluoride. My work in Smith’s Lab involved studies on periodate oxidation products of glycosides. On receiving my PhD I stayed on as a postdoctoral fellow and instructor for two additional years. Happily for me, I was awarded a Guuggenheim Fellowship which allowed me to spend 2 years in Europe, first with Bill Whelan at the Lister Institute of Preventive Medicine in London, then with Bengt Lindberg in Stockholm. My work in both places involved the isolation, characterization, and chemical and enzymatic synthesis of oligosaccharides from natural sources. These were two wonderful years to work in and experience the scientific and cultural milieu of two different European laboratories. Bill Whelan was an enthusiastic and a helpful mentor. We both smoked at that time, and each morning ‘‘after lighting up,’’ we discussed my research. He always provided insightful and valuable ideas and approaches to pursue. My habit of arising early and getting to the lab in the early morning persisted while in London. At first, this did not go over well with the janitorial staff at the Lister. But after some time we became good friends and they greeted me with much enthusiasm each morning. There was another advantage from arriving early: I had the first use of equipment which was, at times, in short supply. While at the Lister I also had the opportunity of meeting Walter Morgan and his colleague Winifred Watkins. They had been studying the chemistry of human blood group substances for many years and were in a fierce competition with Elvin Kabat, Prof. of Immunochemistry at Columbia University. They were always first to grab a copy of the latest journal to see if Kabat had scooped them. Little did I realize, at the time, that our paths would converge. They did indeed when my lab took up a study of the lima bean lectin which they had used to assay for human type A blood group substance. I had many interesting and fruitful discussions with these colleagues during the following years. My first independent position was at the University of New York at Buffalo. To assist a colleague in the characterization of glycogen from microorganism, I decided to prepare concanavalin A from jack beans (Canavalia ensiformis) which was shown to precipitate glycogen. I followed the procedure of James Sumner who first prepared the protein. I decided to pass the purified protein through Sephadex, crossed-linked dextran, to assess its homogeneity. It did not emerge from the column! In an attempt to displace it from the column I suggested to a graduate student to add glucose. Pure concanavalin A was eluted! Address correspondence to: Irwin J. Goldstein, Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 481095606, USA. E-mail: igoldste@umich.edu Received 12 September 2009; accepted 12 September 2009

Quantitative determination of lycorine in Amaryllidaceae plants

Lycorine, a widespread alkaloid in Amaryllidaceae species, has been proven to have antiviral, cytotoxic and antimalarial activities [1–3]. A reversed-phase high-performance liquid chromatographic method has been developed and validated for the determination of lycorine in Amaryllidaceae plants. A simple method for the extraction of lycorine in low-mass plant samples was employed utilizing pre-packed columns with diatomaceous earth (Extrelut®) [4]. The chromatographic separation was performed using an isocratic system with a mobile phase trifluoroacetic acid-water-acetonitrile (0.01:90:10) and diode array detector [5]. The linearity of the method was studied by injecting five known concentrations of the standard in the range of 62.5–500µgmL-1. The calibration curve was determined as Y=23.9788285 X + 62.391901. Validation procedures showed that the method was specific, accurate and precise.

Cloning and Heterologous Expression of a New 3ʹ-Hydroxylase Gene from Lycoris radiata

A full-length cDNA (LC3'H) was obtained from a cDNA library of Lycoris radiata by DOP-PCR (degenerate oligonucleotide primer PCR), 3'race and 5'race methods. Compared with the other reported enzymes from different plants, the deduced amino acid sequence of LC3'H exhibits significant homologies to 3'-hydroxylases that are involved in the caffeic acid biosynthesis. These findings suggest that the new gene is closely related to the biosynthesis of caffeic acid, which is also an important step of the galanthamine biosynthesis in Amaryllidaceae plants.

ALZHEIMER DISEASE AND ACETYLCHOLINESTERASE INHIBITORS

Alzheimer’s disease (AD) or Alzheimer’s is a is a chronic neurodegenerative disorder. It’s characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions. AD patients are identified by problems with memory and language, getting lost and desorientation. AD affects about 6% of people 65 years and older but 4% to 5% of cases are younger.1 It is one of the most costly diseases in the world. In 2015, there were about 48 million people worldwide with AD. To prevent AD, intellectual activities such as reading and playing chess are recommended. In muslim countries, learning quran is linked to a reduced risk of AD. The cause for most Alzheimer’s cases is still mostly unknown except for 1% to 5% of cases linked to genetic factors AD has been identified as a protein misfolding disease (proteopathy), caused by plaque accumulation of abnormally folded amyloid beta protein in the brain.2 However, there is another hypothesis which proposes that AD is caused by reduced synthesis of the neurotransmitter acetylcholine.3 The enzyme Acetylcholinesterase breaks acetylcholine down after acetylcholine is used in the brain. Galanthamine, a natural product isolated from Galanthus nivalis, G. woronowii and some other Amaryllidaceae plants and rivastigmine which is a natural derived compound and Donepezil which is a synthetic compound are used as cholinesterase inhibitors. Polyphenols which have been shown to possess antioxidant activity are the principal constituants of the mediterranean diet which is known to decrease the risk of AD. Antioxidants may slow the progression of AD and minimise neuronal degeneration.4 The compounds that exhibit anticholinesterase activity are also related to radical scavenging activity.5 Polyphenols and terpenoids from Lamiaceae plants possess antioxidant and anticholinesterase activities

Anticancer evaluation of structurally diverse Amaryllidaceae alkaloids and their synthetic derivatives

Plants of the Amaryllidaceae family have been under intense scrutiny for the presence of the specific metabolites responsible for the medicinal properties associated with them. The study began in 1877 with the isolation of alkaloid lycorine from Narcissus pseudonarcissus and since then more than 100 alkaloids, exhibiting diverse biological activities, have been isolated from the Amaryllidaceae plants. Based on the present scientific evidence, it is likely that isocarbostyril constituents of the Amaryllidaceae, such as narciclasine, pancratistatin and their congeners, are the most important metabolites responsible for the therapeutic benefits of these plant species in the folk medical treatment of cancer. Notably, Narcissus poeticus L., used by the ancient Greek physicians, is now known to contain about 0.12 g of narciclasine per kg of fresh bulbs. The focus of the present research work is the chemistry and biology of these compounds as specifically relevant to their potential use in medicine. In particular, the anticancer evaluation of lycorine, narciclasine as well as of other Amaryllidaceae alkaloids and their synthetic derivatives are presented in this paper. The structure–activity relationships among some groups of Amaryllidaceae alkaloids will be discussed.

Two New Amaryllidaceae Alkaloids from the Bulbs of Lycoris radiata

Two new Amaryllidaceae alkaloids, named lycoranines A (1) and B (2), were isolated from the bulbs of Lycoris radiata. Their structures were elucidated on the basis of extensive spectroscopic analysis. Compound 2 was a new-type alkaloid, which provided a new insight into the biosynthesis of alkaloids in Amaryllidaceae plants.

Influence of plant origin on propagation capacity and alkaloid biosynthesis during long-term in vitro cultivation of Leucojum aestivum L.

The investigation deals with in vitro clonal propagation of L. aestivum L. (summer snowflake), a threatened Amaryllidaceae plant species in Bulgaria used in the pharmaceutical industry as raw material for production of galanthamine-based medicines. Plants of known origin and with different alkaloid profile were taken from the living collection of the Institute of Botany, Sofia. Bulbs were used to initiate in vitro cultures and 24 clones were multiplied. The influence of the clone origin on the propagation coefficient, shoot and bulblet morphology, alkaloid profile and content of galanthamine, lycorine, and four related alkaloids was evaluated. Clones kept stable alkaloid profiles and for most of them, high regeneration rates were noted. Galanthamine content of some clones was commensurable with that of Bulgarian populations of L. aestivum of commercial importance. Five clones: four galanthamine-type and one lycorine-type were selected as promising for further investigation.

Quantitative analysis of lycorine in Sternbergia species growing in Turkey

Plants of the family Amaryllidaceae are well known not only for their ornamental value but also for the alkaloids they produce. Some of these alkaloids exhibit interesting pharmacological and/or biological properties. However, the most extensively studied effects are those of non-specific inhibition, such as antiviral and antitumour activities [1]. Sternbergia species, a member of this family, was found to contain lycorine as a major alkaloid. Sternbergia is represented by 6 taxa in Turkey [2].

Analysis of galanthamine‐type alkaloids by capillary gas chromatography–mass spectrometry in plants

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.

New rapid validated HPTLC method for the determination of galanthamine in Amaryllidaceae plant extracts

The work reported in this paper aims at developing an accurate, specific, repeatable and robust HPTLC method for the determination of galanthamine in different Amaryllidaceae plant extracts.

Chemistry, biology, and medicinal potential of narciclasine and its congeners.

Ornamental flower growers know that placing a cut daffodil (a.k.a. narcissus) in a vase with other flowers has a negative effect on the quality of those flowers and significantly shortens their vase life. Furthermore, a common horticultural practice for the cultivation of narcissus flowers involves the introduction of cuts on the bulbs before immersing them into water. The mucilage that leaches out from the cuts is constantly removed by frequent changing of water and this leads to sprouting. These observations raise speculation that specific components in the mucilage of the narcissus bulbs may have powerful growth-inhibitory effects. Historical use of narcissus flowers, as well as at least thirty other plants of the Amaryllidaceae family, in folk medicine for the management of cancer1 speaks volumes to validate this conjecture. Indeed, powerful anticancer properties of Narcissus poeticus L. were already known to the Father of Medicine, Hippokrates of Kos (ca. B.C. 460–370), who recommended a pessary prepared from narcissus oil for the treatment of uterine tumors.2 His successors, the ancient Greek physicians Pedanius Dioscorides (ca. A.D. 40–90) and Soranus of Ephesus (A.D. 98–138) continued using this therapy in the first and second centuries A.D.3,4 In addition, the topical anticancer uses of extracts from this plant5,6 as well as from N. pseudonarcissus7–9 were recorded in the first century A.D. by the Roman natural philosopher Gaius Plinius Secundus, (A.D. 23–79), better known as Pliny the Elder.10 Even the Bible provides multiple references to the Mediterranean N. tazetta L., which has a long history of use against cancer.11 The applications of narcissus oil in cancer management continued in the middle ages in Chinese, North African, Central American and Arabian medicine.1,12 The uses of other genera of the Amaryllidaceae family were also common, e. g. Hymenocallis caribaea (L. emend Gawler) Herbert, utilized by early European medical practitioners for inflammatory tumors.13 More recently, the plants of the Amaryllidaceae have been under intense scrutiny for the presence of the specific metabolites responsible for the medicinal properties associated with this plant family. The study began in 1877 with the isolation of alkaloid lycorine from Narcissus pseudonarcissus14 and since then more than 100 alkaloids, exhibiting diverse biological activities, have been isolated from the Amaryllidaceae plants. Based on the present scientific evidence, it is likely that isocarbostyril constituents of the Amaryllidaceae, such as narciclasine, pancratistatin and their congeners, are the most important metabolites responsible for the therapeutic benefits of these plants in the folk medical treatment of cancer. Notably, N. poeticus L. used by the ancient Greek physicians, as was eluded before, is now known to contain some 0.12 g of narciclasine per kg of fresh bulbs.15 Continuing along this intriguing path, the focus of the present review is a comprehensive literature survey and discussion of the chemistry and biology of these compounds as specifically relevant to their potential use in medicine. The examination of the synthetic organic chemistry, more specifically the total synthesis efforts inspired by the challenging chemical structures of narciclasine, pancratistatin and their congeners, will be reduced to a minimum in view of the two very recent excellent reviews published on this subject.16,17

New Rapid Validated HPTLC Method for the Determination of Lycorine in Amaryllidaceae Plants Extracts.

A rapid, accurate, specific, repeatable and robust HPTLC method for the determination of lycorine in different Amaryllidaceae plant extracts is presented in this work. No article related to the HPTLC determination of lycorine in plant extracts has been reported in literature. Lycorine, a common alkaloid of family Amaryllidaceae, moreover, there have been some recent reports which reveal the interaction of lycorine with DNA and tRNA. It has, therefore, been to the interest of phytochemists to determine the content of this alkaloid in Amaryllidaceaous plants.

Avaliação da atividade antiviral e determinação do perfil cromatográfico de Hippeastrum glaucescens (Martius) Herbert (Amaryllidaceae)

Plantas da família Amaryllidaceae são caracterizadas pela presença de alcalóides isoquinolínicos. Desde o primeiro estudo envolvendo alcalóides desta família em 1877, um grande número destas plantas tem sido analisado quimicamente. Estes compostos apresentam uma ampla variedade de atividades biológicas, tais como: antiviral, citotóxica, antitumoral e analgésica. Neste trabalho, foram avaliados o perfil cromatográfico e a potencial atividade antiviral das frações diclorometano A e B, isoladas dos diferentes órgãos vegetais (bulbos, raízes, folhas e flores) de Hippeastrum glaucescens (Martius) Herbert, assim como dos alcalóides licorina, tazetina e pretazetina, previamente isolados desta planta. A extração dos alcalóides de H. glaucescens foi realizada por métodos clássicos, a partir de bulbos, raízes, folhas e flores fornecendo rendimentos totais em alcalóides de 0,53%; 0,81%; 0,29% e 0,12%, respectivamente. Empregando-se cromatografia em camada delgada, verificou-se que os bulbos e as raízes apresentam perfis cromatográficos semelhantes e que os alcalóides licorina, tazetina e pretazetina estão presentes em todas as partes testadas do vegetal. As frações diclorometano A e B, de cada órgão vegetal, e os alcalóides isolados (licorina, tazetina e pretazetina) não inibiram a replicação do herpesvírus simples humano tipo 1 (HSV-1) cepa KOS, quando avaliados através do método de inibição do efeito citopático viral.

Antimalarial activity screening of some alkaloids and the plant extracts from Amaryllidaceae.

Four groups of Amaryllidaceae alkaloids, namely lycorine-, crinine-, tazettine-, and galanthamine-type, as well as plant extracts of the Amaryllidaceae plants (Pancratium maritimum, Leucojum aestivum, and Narcissus tazetta ssp. tazetta) growing in Turkey were evaluated in vitro for their ability to inhibit Plasmodium falciparum growth by a high-throughput screening method with a 96-well microtiter plate. All four groups of alkaloids exhibited antimalarial activity at different potencies. 6-Hydroxyhaemanthamine, haemanthamine and lycorine were found to be the most potent alkaloids against P. falciparum (T9.96) and galanthamine and tazettine had the least potent activity against P. falciparum (K1).

Rapid Method for Determination of Galanthamine in Amaryllidaceae Plants Using HPLC

A method for rapid qualitative and quantitative determination of galanthamine in Amaryllidaceae plants using reversed phase high performance liquid chromatography has been developed. The use of 0.1% trifluoroacetic acid (TFA) in water as the extraction solvent provides an HPLC‐ready sample, which makes this method simple and efficient. The separation was achieved by a system consisting of a reversed phase C18 small pore (100 Å) column and TFA:water:acetonitrile (0.01:95:5 v/v/v) as the mobile phase. Ephedrine is a suitable internal standard.

Bioactivity-Directed Fractionation of Alkaloids from Some Amaryllidaceae Plants and Their Anticholinesterase Activity

Chloroform:methanol extracts of the bulbs of five Amaryllidaceae plants, namely Galanthus elwesii Hooker fil., G. Ikariae L., Narcissus tazetta subsp. tazetta L., Leucojum aestivum L., and Pancratium maritimum L. growing in Turkey, were evaluated for their anticholinesterase activity by the Ellman method in comparison with galanthamine as the standard drug. Bioactivity-directed fractionation and isolation studies carried out on G. ikariae and N. tazetta subsp. tazetta extracts afforded eight Amaryllidaceae-type alkaloids in total. We found that the activity of both plant extracts was due to the synergistic interaction of the alkaloids isolated.

Galantamine: a new treatment for Alzheimer’s disease

Galantamine (Reminyl®) is a tertiary alkaloid, originally isolated from the Caucasian snowdrop and several Amaryllidaceae plants. It has been used for over 40 years in anesthetics to reverse the effects of curarization. In vitro and in vivo studies have confirmed that galantamine is an orally-active, reversible, competitive inhibitor of brain cholinesterase, which is 50 times more selective for acetylcholinesterase, compared with butyrylcholinesterase. Galantamine has also been shown to allosterically potentiate sub-maximal nicotinic responses to acetylcholine. This direct effect on pre- and postsynaptic nicotinic receptors may represent a second mechanism of action for enhancing cholinergic function, but as yet it is unclear as to whether that has any clinical significance. Galantamine has been licensed for use in the symptomatic treatment of Alzheimer’s disease patients, after proving its efficacy in a number of well-designed clinical trials. It has to be administered twice daily and has shown good tolerability, which typically for drugs of this class, is improved by slow titration. The most common side effects are: nausea, vomiting, diarrhea and anorexia. Galantamine appears to be an effective treatment for Alzheimer’s disease comparable with the other acetylcholinesterase inhibitors in improving cognition and function with growing evidence for its effects on behavior and caregiver burden.

Review of the acetylcholinesterase inhibitor galanthamine

Galanthamine (or galantamine, Reminyl®) is a tertiary alkaloid acetylcholinesterase inhibitor (AChEI) which has been approved in several countries for the symptomatic treatment of senile dementia of the Alzheimer’s type. Derived from bulbs of the common snowdrop and several Amaryllidaceae plants, (-)-galanthamine (GAL) has long been used in anaesthetics to reverse neuromuscular paralysis induced by turbocurarine-like muscle relaxants and more recently, has been shown to attenuate drug- and lesion-induced cognitive deficits in animal models of learning and memory. GAL directly inhibits acetylcholinesterase activity, while demonstrating much weaker activity on butyrylcholinesterase (BuChE). GAL also stimulates pre- and postsynaptic nicotinic receptors, although the clinical significance of this finding is yet unclear. Numerous variants and analogues of GAL have also been developed, with varying potency in inhibiting AChE activity. GAL is readily absorbed after oral administration, with a tmaxof 52 min and a plasma elimination t½ of 5.7 h. The efficacy of GAL administered to Alzheimer’s disease (AD) patients has been well demonstrated by large-scale clinical trials. Typical of AChEIs, the most common adverse events associated with GAL are nausea and vomiting. In conclusion, evidence to date suggests galanthamine to be similar to other AChEIs in improving cognitive function in AD patients.

Effect of sucrose on growth and galanthamine production in shoot-clump cultures of Narcissus confusus in liquid-shake medium

In order to produce galanthamine, an alkaloid currently being tested in Alzheimer's disease therapy, we have used in vitro organ cultures of Narcissus confusus (Amaryllidaceae) plants starting from two different explants: double scale segments with basal plate from bulbs (organogenic cultures), and mature seeds (callogenic-organogenic cultures). Shoot-clumps were induced from buds obtained from twin-scales and from organogenic calluses on a MS medium supplemented with 1 mg l−1 2,4-D and 5 mg l−1 BA. Shoot-clumps were then developed partially submerged in a liquid medium. After one month of precondition, the shoot-clumps were cultured in liquid media with different concentrations of sucrose, from 3% to 18% (w/v) for 14 days. The growth of the regenerated plants treated with 9% sucrose was significantly greater. Under a photoperiod 16 h light/8 h dark, the shoot-clump cultures subjected to the two highest sucrose concentrations gave rise to higher dry weight/fresh weight ratios. Different doses of sucrose affected not only the alkaloid profile in the shoot-clump tissues but also that excreted to the medium. In all cases, shoot cultures of N. confusus were capable of galanthamine biosynthesis, with the best results at 9% sucrose concentration.

Purification and Characterization of a Mannose-Specific Lectin from Shallot (Allium ascalonicum) Bulbs

A new mannose-binding lectin was isolated from shallot (Allium ascalonicum) bulbs by affinity chromatography on an immobilized D-mannose column. The lectin (A. ascojonicum agglutinin, AAA) appeared homogeneous by polyacrylamide gel electrophoresis at pH 4.3 and gave a single protein band with an apparent Mr of 11 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single symmetrical peak of 11 kDa by gel filtration oa a Sephacryl S-200 HR column, indicating that AAA exists as a monomeric protein at neutral pH under the gel filtration condition employed. However, chemical cross-linking studies revealed that some degree of self-association of the lectin molecules occurs and that the lectin exists in solution as a mixture of monomers and oligomers. Scatchard analysis of equilibrium dialysis data showed the presence of one carbohydrate binding site for Man (α1-3) Man-α-O-Me per monomer, with Ka = 1.62 × 104 M−1. The carbohydrate-binding properties of the purified AAA were investigated by quantitative precipitation and hapten inhibition assays. Purified AAA precipitated asialofetuin, asialotransferrin, asialothyroglobulin, asialoorosomucoid, as well as their agalacto derivatives, but did not precipitate either sialylated glycoproteins or mucins. AAA also reacted strongly with the highly branched yeast mannan obtained from Saccharomyces cerevisiae. Of the monosaccharides tested only D-mannose was a hapten inhibitor of the AAA-asialofetuin precipitation system, whereas D-glucose, D-altrose, D-talose, N-acetyl-D-mannosamine, and derivatives of D-mannose, including 2-deoxy-, 2-deoxy-2-fluoro-, 3-deoxy-, and 6-deoxy-D-mannose were noninhibitors. These results suggest that the presence of equatorial hydroxyl groups at the C-3 and C-4 positions, an axial hydroxyl group at the C-2 position, and a free hydroxyl group at the C-6 position of the pyranose ring are the most important loci for the binding of D-mannose to AAA. Of the oligosaccharides tested, the best inhibitors were oligosaccharides containing terminal Man(α1-6)[Man(α1-3)]Man groups. Oligosaccharides containing either Man(α1-3)Man or Man(α1-6)Man units were also moderately good inhibitors of the AAA-asialofetuin precipitation system. These results indicate that AAA has an extended carbohydrate-binding site, which is most complementary to a branched mannotriosyl residue, i.e., Man(α1-6)[Man(α1-3)]Man. A comparison is presented of the detailed carbohydrate-binding properties and molecular structure of the A. ascalonicum lectin and several other mannose-binding lectins from different species of the plant family Amaryllidaceae, i.e., snowdrop lectin (Galanthus nivalis), daffodil lectin (Narcissus pseudonarcissus), and amaryllis lectin (Hippeastrum hybr.).