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The AChE inhibitory activity of alkaloid extracts and compounds has been in the focus of research on the plants of Amaryllidoideae subfamily since the approval of galanthamine in 2001 by FDA for treatment of mild to moderate Alzheimer's disease. A small fraction of the huge biodiversity of the plants producing Amaryllidaceae alkaloids has been studied as a source of AChE inhibitors. Less than 20% of the known Amaryllidaceae alkaloids have been tested in vitro for their ability to inhibit AChE. Galanthamine, lycorine and haemanthamine are scaffolds for semi-synthesis of potent AChE inhibitors. In the last years, galanthamine has been studied in silico for drug optimization and synthesis of potent dual-binding AChE inhibitors. In silico studies of other Amaryllidaceae alkaloids have also provoked the interest of researchers in the last years. The aim of this article is to provide a comprehensive review on studies of the AChE inhibitory activity of extracts and compounds from the plants of Amaryllidoideae subfamily covering the literature from the last two decades.

Cliniatines A–C (1–3), three new Amaryllidaceae alkaloids, consisting of 2,6-dimetylpyridine and lycorine-type and/or galanthamine-type were isolated from Clivia miniata (Lindl.) Bosse. The structures and absolute configurations of 1–3 were elucidated based on spectroscopic data and chemical correlation. Cliniatines A–C showed moderate inhibitory activity against acetylcholinesterase.

Leucojum aestivum is known for its ability to biosynthesize alkaloids with therapeutic properties, among which galanthamine used for the treatment of Alzheimer's disease. New sources of this alkaloid are still being explored. In this study, a novel strain PLV of endophytic bacterium Paenibacillus lautus was isolated from in vitro L. aestivum plants. We report the whole genome sequence of that strain and its capacity to produce alkaloids and growth regulators. The effect of elicitation with autoclaved bacteria on the production of alkaloids was examined. Ten alkaloids were identified in bacteria extracts: galanthamine, lycorine, ismine, lycoramine, haemanthamine, tazettine, galanthine, homolycorine, 1,2-dihydrochlidanthine, and hippeastrine. The mean contents of galanthamine and lycorine were 37.51 µg/g of dry weight (DW) and 129.93 µg/g of DW, respectively. Moreover, isolated P. lautus strain synthesized: indole-3-acetic acid, t-zeatin, c-zeatin, kinetin, gibberellin A1, abscisic acid, salicylic acid, benzoic acid. In vitro elicitation of cultures with P. lautus increased dry biomass, stimulated galanthamine and lycorine production, contributed to 8,9-desmethylenebis (oxy)-7,9 dimethoxy-crinan biosynthesis, change pigments content, and antioxidant enzymes activities. Our findings for the first time point out that galanthamine can be synthesized by an microorganism. Moreover isolated strain can be used as a new elictor of Amaryllidaceae alkaloids biosynthesis.

Lycoris longituba is an important ornamental and medicinal plant and contains various Amaryllidaceae alkaloids such as galanthamine (GAL), lycorine (LYC), and lycoramine (LYCM), which have been reported to exhibit medicinal values. However, the effects of light quality on L. longituba are unknown. The present study aimed to explore the effects of different light emitting diode (LED) light quality on growth, physiological characteristics, and alkaloid accumulations in the seedlings. Lycoris longituba seedlings were cultured under different ratios of blue and red LEDs. The results showed that compared to CK, RB (1:2) treatment improved growth, biomass accumulation, and the synthesis of photosynthetic pigments and reduced membrane lipid peroxidation. Blue light increased GAL, LYC, and LYCM contents which were 2.45-, 1.74-, and 1.92-fold higher than CK, respectively. Most of Amaryllidaceae alkaloids biosynthetic pathway genes including PAL, C4H, NBS, TYDC, OMT, and CYP96T1 were upregulated under B treatment. In general, RB (1:2) was the optimal light quality condition for the growth of L. longituba seedlings. B treatment could promote the accumulation of alkaloids and related gene expressions. This study has established the theoretical foundation and technical support for the use of LED light quality control technology in the production of high-quality L. longituba seedlings.

Two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry were performed to compare the proteomic alterations of lycorine-treated and control cells to further investigate the anti-multiple myeloma (MM) mechanisms of lycorine. Mass spectrometry results showed that after lycorine treatment of MM cells, 42% of the differentially expressed proteins had subcellular localization, mainly, on mitochondria. Voltage-dependent anion-selective channel protein 2 (VDAC2), the most abundant protein in the outer mitochondrial membrane, was up-regulated after treatment with lycorine and was subsequently verified by western blot analysis. Further studies on mitochondria found that lycorine was able to increase abnormal mitochondria and increase mitochondrial membrane potential. Lycorine can achieve the effect of resisting multiple myeloma by acting on VDAC2 and causing mitochondrial abnormalities.

Amaryllidaceae alkaloids are a group of specialized metabolites found predominantly in the Amaryllidaceae plant family. Approximately 600 naturally occurring Amaryllidaceae alkaloids have been identified, many of which possess a variety of potent biological and pharmacological properties. However, only the Amaryllidaceae alkaloid galanthamine, an acetylcholinesterase inhibitor used to treat symptoms of neurodegenerative disorders such as Alzheimer’s disease, is currently on the market. Despite promising pharmacological potential, the clinical development of Amaryllidaceae alkaloids is hindered by limited commercial availability. In contrast to the large body of knowledge on the pharmacological and phytochemical aspects of Amaryllidaceae alkaloids, their molecular and physiological features are less explored. A better understanding of Amaryllidaceae alkaloid biosynthesis and metabolic regulation is crucial to take advantage of new metabolic engineering technologies for improving the efficiency and sustainability of plant or microbial Amaryllidaceae alkaloid production. Although there is still much to learn, over the past several years comparative transcriptomic and traditional biochemical approaches have led to significant advances in the identification of the molecular players involved in producing Amaryllidaceae alkaloids including enzymes from the shikimate and phenylpropanoid pathways. The identity of few Amaryllidaceae alkaloid biosynthetic enzymes and several genes encoding them has been reported. In this review, an overview of the current knowledge on the biosynthesis of Amaryllidaceae alkaloids is presented.

Plants of the Amaryllidaceae family, have a wide distribution through both tropical and sub-tropical regions of the world. They have also a long and notable place in the history of traditional and Western medicine. Amaryllidaceae alkaloids are a structurally diverse group of plant metabolites with a wide range of biological properties including antimalarial, antitumor, antimicrobial, antiviral, enzyme inhibitory, antioxidant, anticonvulsant and antifungal activities. Among them, the haemanthamine-type alkaloids, which have the 5,10b-ethanophenanthridine skeleton as the core structure, represented by haemanthamine and haemanthidine, have received considerable attention, since they have been reported to possess antitumor properties. The present review aims to summarize comprehensively the research that has been published on the Amaryllidaceae alkaloids haemanthamine and haemanthidine.

Tyrosine decarboxylase (TyDC), a pyridoxal-5′-phosphate (PLP)-dependent aromatic amino acid decarboxylase, catalyzes the decarboxylation of l-tyrosine (Tyr) to generate the important biogenic tyramine. This amine was reported to be a biosynthetic precursor of the Amaryllidaceae alkaloids, a group of pharmaceutically important natural products. Herein, we cloned two TyDC-encoding genes from Lycoris radiata, a galanthamine (GAL, a characteristic Amaryllidaceae alkaloid)-producing and medicinally and ornamentally important perennial herbaceous plant. Heterologous expression of LrTyDCs in Escherichia coli and the following purification gave recombinant LrTyDCs to homogeneity for enzymatic reactions. Tyramine was detected and validated by HPLC–DAD and HRESIMS analyses of the LrTyDC-catalyzed reaction mixture, which revealed the catalytic decarboxylation activity of LrTyDCs. The transcriptional LrTyDCs were detected in all tissues of L. radiata by quantitative real-time PCR analyses. The present works demonstrated that TyDC-catalyzed decarboxylation reaction is a key step to generate the non-catechol biosynthetic precursor of the Amaryllidaceae alkaloids.

Fifteen Amaryllidaceae alkaloids (1–15) of various structural types were isolated by standard chromatographic methods from fresh bulbs of Narcissus poeticus cv. Pink Parasol. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic analyses, and by comparison with literature data. Narcipavline (5) and narcikachnine (6) are reported here for the first time. In their structure are combined two basic structural types of Amaryllidaceae alkaloids (galanthamine- and galanthindole-structural types), which represent a new structural type of these compounds. Alkaloids isolated in sufficient amounts were evaluated for their human erythrocytic acetylcholinesterase, and human serum butyrylcholinesterase (HuBuChE) inhibition activity using Ellman’s method. Z-Gly-Pro-p-nitroanilide was used as substrate in the prolyl oligopeptidase (POP) assay. Untested alkaloids were also screened for their cytotoxic activity against a small panel of human cancer cells, which spanned cell lines from different tissue types. In parallel, MRC-5 human fibroblasts were employed to determine overall toxicity against noncancerous cells. Some compounds were evaluated for their antiprotozoal activity. The newly isolated alkaloid narcipavline (5) showed interesting HuBuChE inhibition activity (IC50 = 24.4 ± 1.2 µM), and norlycoramine (11) demonstrated promising POP inhibition (IC50 = 0.21 ± 0.01 mM).

Although the potential of plants extracts to improve feed efficiency and animal productivity and decrease methane emissions by enteric fermentation has been shown, the information available is often contradictory which has been attributed to differences in the complex mixture of bioactive compounds and their interactions. Understanding the degree to which structural features in a compound may affect the biological activity of an extract is essential. We hypothesised that relative small variations in the structure of a compound can have a significant influence on the ability of the derivatives to alter fermentation in the rumen. Nine compounds were synthetized from the natural alkaloid haemanthamine and tested in vitro for their effects on rumen protozoa and fermentation parameters. Our results showed that simple esterifications of haemanthamine or its derivative dihydrohaemanthamine with acetate, butyrate, pivalate or hexanoate led to compounds that differed in their effects on rumen fermentation.

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.

Histochemical staining and RNA-seq data demonstrated that the ROS- and plant hormone-regulated stress responses are the key early events of narciclasine signaling in Arabidopsis root cells. Narciclasine, an amaryllidaceae alkaloid isolated from Narcissus tazetta bulbs, employs a broad range of functions on plant development and growth. However, its molecular interactions that modulate these roles in plants are not fully understood. To elucidate the global responses of Arabidopsis roots to short-term narciclasine exposure, we first measured the accumulation of H2O2 and O2 – with histochemical staining, and then profiled the gene expression pattern in Arabidopsis root tips treated with 0.5 µM narciclasine across different exposure times by RNA-seq. Physiological measurements showed a significant increase in H2O2 began at 30–60 min of narciclasine treatment and O2 – accumulated by 120 min. Compared with controls, 236 genes were upregulated and 54 genes were downregulated with 2 h of narciclasine treatment, while 968 genes were upregulated and 835 genes were downregulated with 12 h of treatment. The Gene Ontology analysis revealed that the differentially expressed genes were highly enriched during oxidative stress, including those involved in the “regulation of transcription”, “response to oxidative stress”, “plant–pathogen interaction”, “ribonucleotide binding”, “plant cell wall organization”, and “ribosome biogenesis”. Moreover, Kyoto Encyclopedia of Genes and Genomes pathway enrichment statistics suggested that carbohydrate metabolism, amino acid metabolism, amino sugar and nucleotide sugar metabolism, and biosynthesis of phenylpropanoid and secondary metabolites were significantly inhibited by 12 h of narciclasine exposure. Hence, our results demonstrate that hormones and H2O2 are important regulators of narciclasine signaling and help to uncover the factors involved in the molecular interplay between narciclasine and phytohormones in Arabidopsis root cells.

Background Lycorine has been revealed to inhibit the development of many kinds of malignant tumors, including glioblastoma multiforme (GBM). Although compelling evidences demonstrated Lycorine’s inhibition on cancers through some peripheral mechanism, in-depth mechanism studies of Lycotine’s anti-GBM effects still call for further exploration. Epidermal Growth Factor Receptor (EGFR) gene amplification and mutations are the most common oncogenic events in GBM. Targeting EGFR by small molecular inhibitors is a rational strategy for GBM treatment. Methods The molecular docking modeling and in vitro EGFR kinase activity system were employed to identify the potential inhibitory effects of Lycorine on EGFR. And the Biacore assay was used to confirm the direct binding status between Lycorine and the intracellular EGFR (696–1022) domain. In vitro assays were conducted to test the suppression of Lycorine on the biological behavior of GBM cells. By RNA interference, EGFR expression was reduced then cells underwent proliferation assay to investigate whether Lycorine’s inhibition on GBM cells was EGFR-dependent or not. RT-PCR and western blotting analysis were carried out to investigate the underlined molecular mechanism that Lycorine exerted on EGFR itself and EGFR signaling pathway. Three different xenograft models (an U251-luc intracranially orthotopic transplantation model, an EGFR stably knockdown U251 subcutaneous xenograft model and a patient-derived xenograft model) were performed to verify Lycorine’s therapeutic potential on GBM in vivo. Results We identified a novel small natural molecule Lycorine binding to the intracellular EGFR (696–1022) domain as an inhibitor of EGFR. Lycorine decreased GBM cell proliferation, migration and colony formation by inducing cell apoptosis in an EGFR-mediated manner. Furthermore, Lycorine inhibited the xenograft tumor growths in three animal models in vivo. Besides, Lycorine impaired the phosphorylation of EGFR, AKT, which were mechanistically associated with expression alteration of a series of cell survival and death regulators and metastasis-related MMP9 protein. Conclusions Our findings identify Lycorine directly interacts with EGFR and inhibits EGFR activation. The most significant result is that Lycorine displays satisfactory therapeutic effect in our patient-derived GBM tumor xenograft, thus supporting the conclusion that Lycorine may be considered as a promising candidate in clinical therapy for GBM.

Amaryllidaceae alkaloids are an example of the vast diversity of secondary metabolites with great therapeutic promise. The identification of novel compounds in this group with over 300 known structures continues to be an area of active study. The recent identification of norbelladine 4′-O-methyltransferase (N4OMT), an Amaryllidaceae alkaloid biosynthetic enzyme, and the assembly of transcriptomes for Narcissus sp. aff. pseudonarcissus and Lycoris aurea highlight the potential for discovery of Amaryllidaceae alkaloid biosynthetic genes with new technologies. Recent technical advances of interest include those in enzymology, next generation sequencing, genetic modification, nuclear magnetic resonance spectroscopy (NMR), and mass spectrometry (MS).

Amaryllidaceae alkaloids (AAs) represent a diverse class of plant specialized metabolites and many display potent pharmacological activities. The AA metabolic pathway is poorly understood and resources are minimal. To enable AA pathway elucidation and novel biosynthetic enzymes discovery, we generated comprehensive metabolomic and corresponding transcriptomic datasets from different tissues of Narcissus pseudonarcissus ‘King Alfred’. In this study, we performed untargeted UPLC-QTOF-MS metabolite analysis from different tissues, which generated exhaustive list of compounds, including several AAs, most predominant and diverse in bulbs. RNA sequencing of N. pseudonarcissus ‘King Alfred’ bulbs yielded 195,347 transcripts, after assembly. Top expressed genes belong to process like metabolism, survival, and defense including alkaloid biosynthetic genes. The transcriptome contained complete sequences for all proposed genes encoding AA-biosynthetic enzymes such as tyrosine decarboxylase (TYDC1 and TYDC2), phenylalanine ammonia-lyase (PAL1 and PAL2) and phenolic acids hydroxylases (C4H and C3H) to name a few. Furthermore, transcriptome data were validated using RT-qPCR analysis and expression study in different tissues of N. pseudonarcissus ‘King Alfred’ was performed. Here, we present the first comprehensive metabolome and transcriptome study from N. pseudonarcissus ‘King Alfred’ providing invaluable resources for metabolic engineering and biotechnological applications.

In 1906, Alois Alzheimer, a psychiatrist from Munich, Germany, described a new mental illness, which is a progressively damaging brain disorder, leading to neurodegeneration, erosion of basic intellectual functions along with histological changes, and ultimately the patient’s death. Up until now, and despite enormous efforts in academia and the pharmaceutical industry, there is only modest symptomatic treatment available, but no intervention at the roots of the disease. One of the contemporary active pharmaceutical ingredients applied for this purpose is galantamine, an amaryllidaceae alkaloid, found, e.g., in Galanthus nivalis. In 1960, its correct structure, an amino acid-derived tetracycle, has been established by Shigeru Kobayashi and Sir Derek Barton. Since then, a plethora of syntheses has been published. On industrial scale, galantamine is extracted from the bulbs of daffodils, though it can also be manufactured by total synthesis.

Enhanced mitochondrial stability and decreased dependence on oxidative phosphorylation confer an acquired resistance to apoptosis in cancer cells, but may present opportunities for therapeutic intervention. The compound pancratistatin (PST) has been shown to selectively induce apoptosis in cancer cells. However, its low availability in nature has hindered its clinical advancement. We synthesized PST analogs and a medium-throughput screen was completed. Analogs SVTH-7, -6, and -5 demonstrated potent anti-cancer activity greater than PST and several standard chemotherapeutics. They disrupted mitochondrial function, activated the intrinsic apoptotic pathway, and reduced growth of tumor xenografts in vivo. Interestingly, the pro-apoptotic effects of SVTH-7 on cancer cells and mitochondria were abrogated with the inhibition of mitochondrial complex II and III, suggesting mitochondrial or metabolic vulnerabilities may be exploited by this analog. This work provides a scaffold for characterizing distinct mitochondrial and metabolic features of cancer cells and reveals several lead compounds with high therapeutic potential.

Four new Amaryllidaceae alkaloids (1-4) possessing a homolycorine-type or a crinine-type skeleton have been isolated from the aerial part of Zephyranthes candida, and their structures were elucidated on the basis of spectroscopic data. The stereochemistry was elucidated by combination of NOESY correlations and CD analyses.

Narciclasine (NCS) is an Amaryllidaceae alkaloid isolated from Narcissus tazetta bulbs. Its phytotoxic effects on plant growth were examined in lettuce (Lactuca sativa L.) seedlings. Results showed that high concentrations (0.5-5 μM) of NCS restricted the growth of lettuce roots in a dose-dependent manner. In NCS-treated lettuce seedlings, the following changes were detected: reduction of mitotic cells and cell elongation in the mature region, inhibition of proliferation of meristematic cells, and cell cycle. Moreover, comet assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay indicated that higher levels NCS (0.5-5 μM) induced DNA damage in root cells of lettuce. The decrease in meristematic cells and increase in DNA damage signals in lettuce roots in responses to NCS are in a dose-dependent manner. NCS-induced reactive oxygen species accumulation may explain an increase in DNA damage in lettuce roots. Thus, the restraint of root growth is due to cell cycle arrest which is caused by NCS-induced DNA damage. In addition, it was also found that NCS (0.5-5 μM) inhibited the root hair development of lettuce seedlings. Further investigations on the underlying mechanism revealed that both auxin and ethylene signaling pathways are involved in the response of root hairs to NCS.

Screening of small-molecule libraries is an important aspect of probe and drug discovery science. Numerous authors have suggested that bioactive natural products are attractive starting points for such libraries because of their structural complexity and sp(3)-rich character. Here, we describe the construction of a screening library based on representative members of four families of biologically active alkaloids (Stemonaceae, the structurally related cyclindricine and lepadiformine families, lupin and Amaryllidaceae). In each case, scaffolds were based on structures of the naturally occurring compounds or a close derivative. Scaffold preparation was pursued following the development of appropriate enabling chemical methods. Diversification provided 686 new compounds suitable for screening. The libraries thus prepared had structural characteristics, including sp(3) content, comparable to a basis set of representative natural products and were highly rule-of-five compliant.

Three new alkaloids, 2α-hydroxy-6-O-n-butyloduline, O-n-butyllycorenine, (−)-N-(chloromethyl)lycoramine (1–3), and a new phenolic compound, ((7S)-7-(4-hydroxyphenyl)-7-hydroxypropyl)-2′-methylbenzene-3′,6′-diol (14), along with ten known alkaloids (4–13), were isolated from the bulbs of Lycoris aurea collected from Huaihua County of Hunan Province, China. Their structures were elucidated by spectroscopic methods including HRESIMS, UV, IR, and NMR. All the isolated compounds were tested for their neuroprotective effects against CoCl2 and H2O2-induced SH-SY5Y cell death. Compounds 1–7 and 10 exhibited significant neuroprotective effects against CoCl2-induced SH-SY5Y cell injury, while compounds 1–5, 7, 10 and 12 showed obvious neuroprotective effects against H2O2-induced SH-SY5Y cell death.

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.

Aberrant activation of the NOTCH signaling pathway is crucial for the onset and progression of T cell leukemia. Yet recent studies also suggest a tumor suppressive role of NOTCH signaling in acute myeloid leukemia (AML) and reactivation of this pathway offers an attractive opportunity for anti-AML therapies. N-methylhemeanthidine chloride (NMHC) is a novel Amaryllidaceae alkaloid that we previously isolated from Zephyranthes candida, exhibiting inhibitory activities in a variety of cancer cells, particularly those from AML. Here, we report NMHC not only selectively inhibits AML cell proliferation in vitro but also hampers tumor development in a human AML xenograft model. Genome-wide gene expression profiling reveals that NMHC activates the NOTCH signaling. Combination of NMHC and recombinant human NOTCH ligand DLL4 achieves a remarkable synergistic effect on NOTCH activation. Moreover, pre-inhibition of NOTCH by overexpression of dominant negative MAML alleviates NMHC-mediated cytotoxicity in AML. Further mechanistic analysis using structure-based molecular modeling as well as biochemical assays demonstrates that NMHC docks in the hydrophobic cavity within the NOTCH1 negative regulatory region (NRR), thus promoting NOTCH1 proteolytic cleavage. Our findings thus establish NMHC as a potential NOTCH agonist that holds great promises for future development as a novel agent beneficial to patients with AML.

The plant hormone auxin plays a central role in the regulation of plant growth and development, as well as in responses to environmental stimuli. Narciclasine (NCS, an Amaryllidaceae alkaloid) isolated from Narcissus tazetta bulbs has a broad range of inhibitory effects on plants. In this study, the role of NCS in responses to auxin in Arabidopsis thaliana roots was investigated. We demonstrated the inhibitory effects of NCS on auxin-inducible lateral root formation, root hair formation, primary root growth, and the expression of primary auxin-inducible genes in Arabidopsis roots using DR5::GUS reporter gene, native auxin promoters (IAA12::GUS, IAA13::GUS), and quantitative reverse transcription PCR analysis. Results also showed that NCS did not affect the expression of cytokinin-inducible ARR5::GUS reporter gene. NCS relieved the auxin-enhanced degradation of the Aux/IAA repressor modulated by the SCFTIR1 ubiquitin-proteasome pathway. In addition, NCS did not alter the auxin-stimulated interaction between IAA7/AXR2 (Aux/IAA proteins) and the F-box protein TIR1 activity of the proteasome. Taken together, these results suggest that NCS acts on the auxin signaling pathway upstream of TIR1, which modulates Aux/IAA protein degradation, and thereby affects the auxin-mediated responses in Arabidopsis roots.

Phenanthridone alkaloids are envisaged as an attractive lead for the development of anticancer agents. We have prepared a series of aromatized analogues on the basis of the structure of this class of alkaloids with the hope of finding the simplified compounds with comparable activities. The obtained analogues were evaluated for their cytotoxic effect against several cancer cell lines and found to be virtually inactive. These observations together with molecular modeling studies strongly suggest that the stereochemistries of hydroxyl groups in C-ring of phenanthridone alkaloids are crucial to biological effects.

structures, in the present study, we aimed to investigate the alkaloid composition of G. gracilis, collected from a different locality in Turkey, by GC/MS. In this study, the alkaloid extracts of Galanthus gracilis Celak. were examined by means of gas chromatography/mass spectrometry (GC/MS), and 20 alkaloids were reported. Homolycorine and 8-O-demethylhomolycorine are the main alkaloids in the extracts obtained from the aerial parts and bulbs, respectively. Additionally, the AChE inhibitory activity of the extracts prepared from the aerial parts and bulbs of G. gracilis was investigated by in vitro Ellmans method [10]. Potent inhibitory activity was recorded in the extracts of the bulbs and aerial parts of G. gracilis. To the best of our knowledge, this is the first report of a GC/MS study on the alkaloids of G. gracilis growing in Turkey. Based on the GC/MS analysis, a total of 20 alkaloids was detected in two different extracts prepared from G. gracilis (Table 1). The identified compounds possessed various Amaryllidaceae alkaloid skeleton types including phenanthridine, galanthamine, crinine, tazettine, lycorine, and homolycorine. Generally, homolycorine and tazettine type alkaloids were major components in the tested samples. Homolycorine was found to be the main alkaloid in the aerial part extract of G. gracilis (71.62%). Similarly, the extract obtained from the bulbs contained 8-O-demethylhomolycorine (44.39%), homolycorine (18.00%), and galanthindole (17.95%) in very high amounts. Tazettine was found to be present in both of the extracts (aerial parts, 18.41%; bulbs, 6.47%). Moreover, in the extracts of the bulbs, lycorine was detected (5.45%) and galanthamine was found in trace amounts. Due to the absence of related literature, GG-1 and GG-2 were not identified, but their mass fragmentation has specific properties for Amaryllidaceae alkaloids. AChE inhibitory activity potentials of the extracts were carried out using in vitro Ellmans method, and both of the extracts showed significant activity. The bulb extract of G. gracilis (IC 50 11.82 g/mL) was found to be more active than the extract of the aerial parts (IC 50 25.5 g/mL). Galanthamine was used as a positive control (IC 50 0.043 g/mL).

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.

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.

Lycorine is reported to be a multifunctional compound. We previously showed that lycorine is an HCV inhibitor with strong activity. Further research on the antivirus mechanism indicated that lycorine does not affect the enzymes that are indispensable to HCV replication but suppresses the expression of Hsc70 in the host cell to limit HCV replication. However, due to the cytotoxicity and apoptosis induction of lycorine, lycorine is unsafe to be a anti-HCV agent for clinical application. As a result of increasing interest, its structure was optimized for the first time and a novel series of lycorine derivatives was synthesized, all of which lost their cytotoxicity to different degrees. Structure-activity analysis of these compounds revealed that disubstitution on the free hydroxyl groups at C1 and C2 and/or degradation of the benzodioxole group would markedly reduce the cytotoxicity. Furthermore, an α, β-unsaturated ketone would improve the HCV inhibitory activity of lycorine. The C3-C4 double bond is crucial to the anti-HCV activity because hydrogenation of this double bond clearly weakened HCV inhibition.

The alkaloid lycorine, which is considered to inhibit the last step in ascorbic acid biosynthesis, is produced by Narcissus pseudonarcissus. The growth of two strains (C1 and C3) of Cryptococcus laurentii isolated from root tips of N. pseudonarcissus is inhibited by lycorine, as is the in vivo production of ascorbic acid from -galactonic acid-γ-lactone. In contrast, C. laurentii strain C4, isolated from the lycorine-containing bracts of the bulb, was not inhibited by lycorine and did not contain ascorbic acid when cultivated with or without -galactonic acid-γ-lactone.

The phenanthridine alkaloid lycobetaine is a minor constituent of Amaryllidaceae. Inhibition of cell growth was studied in the clonogenic assay on 21 human tumour xenografts (mean IC 50 = 0.8 μM). The growth of human leukaemia cell lines was also potently inhibited (mean IC 50 = 1.3 μM). Athymic nude mice, carrying s.c. implanted human gastric tumour xenograft GXF251, were treated i.p. with lycobetaine for 4 weeks, resulting in a marked tumour growth delay. Lycobetaine was found to act as a specific topoisomerase IIβ poison. In the presence of calf thymus DNA, pure recombinant human topoisomerase IIβ protein was selectively depleted from SDS-gels, whereas no depletion of topoisomerase IIα protein was observed. In A431 cells immunoband-depletion of topoisomerase IIβ was induced, suggesting stabilization of the covalent catalytic DNA-intermediate in living cells. It is reasonable to assume that this mechanism will cause or at least contribute significantly to the antitumour activity. © 2001 Cancer Research Campaign http://www.bjcancer.com

By using lycorine, a specific inhibitor of ascorbate biosynthesis, it was possible to demonstrate that plant cells consume a high quantity of ascorbate (AA). The in vivo metabolic reactions utilizing ascorbate are the elimination of H2O2 by ascorbate peroxidase and the hydroxylation of proline residues present in the polypeptide chains by means of peptidyl-proline hydroxylase. Ascorbate acts in the cell metabolism as an electron donor, and consequently ascorbate free radical (AFR) is continuously produced. AFR can be reconverted to AA by means of AFR reductase or can undergo spontaneous disproportion, thus generating dehydroascorbic acid (DHA). During cell division and cell expansion ascorbate consumption is more or less the same; however, the AA/DHA ratio is 6-10 during cell division and 1-3 during cell expansion. This ratio depends essentially on the different AFR reductase activity in these cells. In meristematic cells AFR reductase is very high, and consequently a large amount of AFR is reduced to AA and a small amount of AFR undergoes disproportionation; in expanding cells the AFR reductase activity is lower, and therefore AFR is massively disproportionated, thus generating a large quantity of DHA. Since the transition from cell division to cell expansion is marked by a large drop of AFR reductase activity in the ER, it is suggested here that AFR formed in this compartment may be involved in the enlargement of the ER membranes and provacuole acidification. DHA is a toxic compound for the cell metabolism and as such the cell has various strategies to counteract its effects: (i) meristematic cells, having an elevated AFR reductase, prevent large DHA production, limiting the quantity of AFR undergoing disproportionation (ii) Expanding cells, which contain a lower AFR reductase, are, however, provided with a developed vacuolar system and segregate the toxic DHA in the vacuole. (iii) Chloroplast strategy against DHA toxicity is efficient DHA reduction to AA using GSH as electron donor. This strategy is usually poorly utilized by the surrounding cytoplasm. DHA reduction does play an important role at one point in the life of the plant, that is, during the early stage of seed germination. The dry seed does not store ascorbate, but contains DHA, and several DHA-reducing proteins are detectable. In this condition, DHA reduction is necessary to form a limited AA pool in the seed for the metabolic requirements of the beginning of germination. After 30-40 h ascorbate ex novo synthesis starts, DHA reduction declines until a single isoform remains, as is typical in the roots, stem, and leaves of seedlings.(ABSTRACT TRUNCATED AT 400 WORDS)

The petite-positive yeast Saccharomyces cerevisiae can be efficiently and completely converted to respiratory-deficient cytoplasmic petite mutants by intercalating drugs. Rho0 petites from Schizosaccharomyces pombe could only be obtained in strains carrying a nuclear mutation. In this paper we report the efficient isolation of rho0 mutants in a Sch. pombe strain containing a mitochondrial mutator mutation. We also show that the alkaloid lycorine is able to differentiate between cells containing defective mitochondrial DNA (mit-) and those lacking mitochondrial DNA completely (rho0). Rho0 cells are resistant to the alkaloid whereas mit- and wild-type cells show the same sensitivity.

Human enterovirus 71 (EV71) infection causes hand, foot and mouth disease in children under 6 years old and this infection occasionally induces severe neurological complications. No vaccines or drugs are clinical available to control EV71 epidemics. In present study, we show that treatment with lycorine reduced the viral cytopathic effect (CPE) on rhabdomyosarcoma (RD) cells by inhibiting virus replication. Analysis of this inhibitory effect of lycorine on viral proteins synthesis suggests that lycorine blocks the elongation of the viral polyprotein during translation. Lycorine treatment of mice challenged with a lethal dose of EV71 resulted in reduction of mortality, clinical scores and pathological changes in the muscles of mice, which were achieved through inhibition of viral replication. When mice were infected with a moderate dose of EV71, lycorine treatment was able to protect them from paralysis. Lycorine may be a potential drug candidate for the clinical treatment of EV71-infected patients.

Background Pancratistatin (PST), a compound extracted from an Amaryllidaceae (AMD) family plant, has been shown to specifically induce apoptosis in cancer cells with no/minimal toxic effect on normal cells. A systematic synthetic approach has indicated that the minimum cytotoxic pharmacophore comprises the trans-fused b/c-ring system containing the 2, 3, 4-triol unit in the C-ring. To further explore the structure-activity relationship of this group of compounds we have investigated the anti-cancer efficacy and specificity of two PST-related natural compounds, AMD4 and AMD5. Both of these compounds lack the polyhydroxylated lycorane element of PST instead having a methoxy-substuituted crinane skeleton. Results Our results indicate that AMD5 has efficacy and selectivity similar to PST, albeit at a 10-fold increased concentration. Interestingly AMD4 lacks apoptotic activity. Conclusion Our results indicate that the phenanthridone skeleton in natural Amaryllidaceae alkaloids may be a significant common element for selectivity against cancer cells; furthermore, the configuration of the methoxy-side groups is responsible for higher binding affinity to the target protein/s thus making for a more efficient anti-cancer agent.

In a previous paper we have shown that the alkaloid lycorine inhibits growth of rho+, mit- and rho-, strains of Saccharomyces cerevisiae, whereas strains devoid of mitochondrial DNA (rho degrees) are resistant to more than 200 micrograms/ml of the alkaloid. In this report we show that hypersuppressive petites are almost as resistant as rho degrees mutants, whereas isogenic rho- petites, which have retained longer segments of the genome, are sensitive to the drug.

Kondos analytische Resultate der Tazettindegradationsprodukte wurden neu bestimmt, Strukturformeln fur diese Substanzen werden vorgeschlagen und die Struktur und Stereochemie des Alkaloids dargestellt.

Mutants resistant to 200 micrograms/ml of the alkaloid lycorine (LYCR) in non-fermentable substrate were isolated after nitrosoguanidine mutagenesis. Tetrad analysis and growth of heterozygous (LYCR/lycS) diploids from two different mutants revealed that a single nuclear and dominant mutation is responsible for the resistant phenotype. In the wild type total protein synthesis is only slightly inhibited, whereas DNA and RNA synthesis is lowered to about 30% of the control. In the lycorine resistant mutants all macromolecular syntheses are unaffected by the drug.