Phylogeny and adaptive evolution of subgenus Rhizirideum (Amaryllidaceae, Allium) based on plastid genomes.

The subgenus Rhizirideum in the genus Allium consists of 38 species worldwide and forms five sections (A. sect. Rhizomatosa, A. sect. Tenuissima, A. sect. Rhizirideum, A. sect. Eduardia, and A. sect. Caespitosoprason), A. sect. Caespitosoprason being merged into A. sect. Rhizomatosa recently. Previous studies on this subgenus mainly focused on separate sections. To investigate the inter-section and inter-subgenera phylogenetic relationships and adaptive evolution of A. subg. Rhizirideum, we selected thirteen representative species, which cover five sections of this subgenus and can represent four typical phenotypes of it. We conducted the comparative plastome analysis with our thirteen plastomes. And phylogenetic inferences with CDSs and complete sequences of plastomes of our thirteen species and another fifty-four related species were also performed. As a result, the A. subg. Rhizirideum plastomes were relatively conservative in structure, IR/SC borders, codon usage, and repeat sequence. In phylogenetic results, the inter-subgenera relationships among A. subg. Rhizirideum and other genus Allium subgenera were generally similar to the previous reports. In contrast, the inter-section relationships within our subgenus A. subg. Rhizirideum were newly resolved in this study. A. sect. Rhizomatosa and A. sect. Tenuissima were sister branches, which were then clustered with A. sect. Rhizirideum and A. sect. Eduardia successively. However, Allium Polyrhizum Turcz. ex Regel, type species of A. sect. Caespitosoprason, was resolved as the basal taxon of A. subg. Rhizirideum. Allium siphonanthum J. M. Xu was also found in clade A. subg. Cyathophora instead of clade A. subg. Rhizirideum. The selective pressure analysis was also conducted, and most protein-coding genes were under purifying selection. At the same time, just one gene, ycf2, was found under positive selection, and another three genes (rbcL, ycf1a, ycf1b) presented relaxed selection, which were all involved in the photosynthesis. The low temperature, dry climate, and high altitude of the extreme habitats where A. subg. Rhizirideum species grow might impose intense natural selection forces on their plastome genes for photosynthesis. In summary, our research provides new insights into the phylogeny and adaptive evolution of A. subg. Rhizirideum. Moreover, we suggest that the positions of the A. subg. Rhizirideum species A. polyrhizum and A. siphonanthum should be reconsidered.

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.

Structurally diverse alkaloids with nine frameworks from Zephyranthes candida and their acetylcholinesterase inhibitory and anti-inflammatory activities.

Twenty-six structurally diverse Amaryllidaceae alkaloids, including ten undescribed compounds named zephyranines A-I and 6-O-ethylnerinine, two undescribed natural products zephyranthine-6-one and 3-O-deacetyl-sternbergine, were isolated from whole plants of Zephyranthes candida. Their structures were determined by HRESIMS, 1D and 2D NMR, CD data analysis, NMR and ECD calculations, and single-crystal X-ray diffraction analysis. All structures were classified into nine framework types: 10b,11-seco-crinine, graciline, crinine, homolycorine, trisphaeridine, lycorine, galasine, tazettine, and belladine. Zephyranine A represents the first naturally occurring 10b,11-seco-crinine type alkaloid, and zephyranine B is the sixth graciline type alkaloid. 6-O-ethylnerinine is an artifact from the extraction and isolation. All isolates were evaluated for their acetylcholinesterase (AChE) inhibitory and anti-inflammatory activities. Zephyranines A, G, and H exhibited moderate AChE inhibitory activities, with IC50 values of 8.2, 39.0, and 10.8 µM, respectively. Zephyranine B, haemanthamine, haemanthidine, 11-hydroxyvittatine, and 8-demethoxy-10-O-methylhostasine exhibited potent anti-inflammatory activity on the LPS-induced NO production in RAW264.7 mouse macrophages with IC50 values of 21.3, 4.6, 12.2, 5.6, and 17.4 µM, respectively. Structure-activity-relationship analysis and docking studies indicated that interactions with the key Trp286 and Tyr337 residues are required for potent AChE inhibitors.

Cliniatines A-C, new Amaryllidaceae alkaloids from Clivia miniata, inhibiting Acetylcholinesterase.

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.

Modelling and measuring complexity of traditional and ancient technologies using Petri nets.

Technologies and their production systems are used by archaeologists and anthropologists to study complexity of socio-technical systems. However, there are several issues that hamper agreement about what constitutes complexity and how we can systematically compare the complexity of production systems. In this work, we propose a novel approach to assess the behavioural and structural complexity of production systems using Petri nets. Petri nets are well-known formal models commonly used in, for example, biological and business process modelling, as well as software engineering. The use of Petri nets overcomes several obstacles of current approaches in archaeology and anthropology, such as the incompatibility of the intrinsic sequential logic of the available methods with inherently non-sequential processes, and the inability to explicitly model activities and resources separately. We test the proposed Petri net modelling approach on two traditional production systems of adhesives made by Ju/'hoan makers from Nyae, Namibia from Ammocharis coranica and Ozoroa schinzii plants. We run simulations in which we assess the complexity of these two adhesive production systems in detail and show how Petri net dynamics reveal the structural and behavioural complexity of different production scenarios. We show that concurrency may be prevalent in the production system of adhesive technologies and discuss how changes in location during the process may serve to control the behavioural complexity of a production system. The approach presented in this paper paves the way for future systematic visualization, analysis, and comparison of ancient production systems, accounting for the inherent complex, concurrent, and action/resource-oriented aspects of such processes.

Chemical Synthesis and Biological Activities of Amaryllidaceae Alkaloid Norbelladine Derivatives and Precursors.

Amaryllidaceae alkaloids (AAs) are a structurally diverse family of alkaloids recognized for their many therapeutic properties, such as antiviral, anti-cholinesterase, and anticancer properties. Norbelladine and its derivatives, whose biological properties are poorly studied, are key intermediates required for the biosynthesis of all ~650 reported AAs. To gain insight into their therapeutic potential, we synthesized a series of O-methylated norbelladine-type alkaloids and evaluated their cytotoxic effects on two types of cancer cell lines, their antiviral effects against the dengue virus (DENV) and the human immunodeficiency virus 1 (HIV-1), and their anti-Alzheimer's disease (anti-cholinesterase and -prolyl oligopeptidase) properties. In monocytic leukemia cells, norcraugsodine was highly cytotoxic (CC50 = 27.0 µM), while norbelladine was the most cytotoxic to hepatocarcinoma cells (CC50 = 72.6 µM). HIV-1 infection was impaired only at cytotoxic concentrations of the compounds. The 3,4-dihydroxybenzaldehyde (selectivity index (SI) = 7.2), 3',4'-O-dimethylnorbelladine (SI = 4.8), 4'-O-methylnorbelladine (SI > 4.9), 3'-O-methylnorbelladine (SI > 4.5), and norcraugsodine (SI = 3.2) reduced the number of DENV-infected cells with EC50 values ranging from 24.1 to 44.9 µM. The O-methylation of norcraugsodine abolished its anti-DENV potential. Norbelladine and its O-methylated forms also displayed butyrylcholinesterase-inhibition properties (IC50 values ranging from 26.1 to 91.6 µM). Altogether, the results provided hints of the structure-activity relationship of norbelladine-type alkaloids, which is important knowledge for the development of new inhibitors of DENV and butyrylcholinesterase.

Amaryllidaceae-Type Alkaloids from Pancratium maritimum: Apoptosis-Inducing Effect and Cell Cycle Arrest on Triple-Negative Breast Cancer Cells.

Aiming to find Amaryllidaceae alkaloids against breast cancer, including the highly aggressive triple-negative breast cancer, the phytochemical study of Pancratium maritimum was carried out. Several Amaryllidaceae-type alkaloids, bearing scaffolds of the haemanthamine-, homolycorine-, lycorine-, galanthamine-, and tazettine-type were isolated (3-11), along with one alkamide (2) and a phenolic compound (1). The antiproliferative effect of compounds (1-11) was evaluated by the sulforhodamine B assay against triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, breast cancer cells MCF-7, and the non-malignant fibroblast (HFF-1) and breast (MCF12A) cell lines. The alkaloids 3, 5, 7, and 11 showed significant growth inhibitory effects against all breast cancer cell lines, with IC50 (half-maximal inhibitory concentration) values ranging from 0.73 to 16.3 µM. The homolycorine-type alkaloid 7 was selected for further investigation in MDA-MB-231 cells. In the annexin-V assay, compound 7 increased cell death by apoptosis, which was substantiated, in western blot analyses, by the increased expression of the pro-apoptotic protein Bax, and the decreased expression of the anti-apoptotic protein Bcl-xL. Consistently, it further stimulated mitochondrial reactive oxygen species (ROS) generation. The antiproliferative effect of compound 7 was also associated with G2/M cell cycle arrest, which was supported by an increase in the p21 protein expression levels. In MDA-MB-231 cells, compound 7 also exhibited synergistic effects with conventional chemotherapeutic drugs such as etoposide.

Complete chloroplast genomes and comparative analyses of Hippeastrum 'milady', Hippeastrum albertii and Hippeastrum reticulatum (Amaryllidaceae).

Hippeastrum is a genus of ornamental plants with large, brightly colored flowers. Due to the very high seed-setting rate of the hybridization of Hippeastrum, the large population of hybrid progeny and the existence of superparent inheritance, it is difficult to trace the origin of the varieties collected from the market during breeding. In this study, we analyzed the chloroplast genomes of Hippeastrum 'Milady', H. alberti, and H. reticulatum using the Illumina NovaSeq sequencing platform and generated full-length sequences of 158,067, 158,067, and 158,522 bp, respectively. All three genomes had the typical tetrad structure. The large single copy, small single copy, and inverted repeat regions of H. reticulatum were observed to be respectively 277, 138, and 20 bp longer than the corresponding regions of H. 'Milady' and H. alberti. The results of comparative analysis of simple sequence repeats (SSRs), Ka/Ks ratios, codon preferences, and complete sequences of chloroplasts of these three taxa and 14 other plant species were as follows. First, the chloroplast genomes of H. 'Milady', H. alberti, and H. reticulatum contain 209, 209, and 211 SSR sites, respectively, most of which (123, 123, and 122, respectively) are single nucleotide repeats. Second, leucine, arginine, and serine are the most frequently used amino acids in the three chloroplast genomes. Third, H. 'Milady', H. alberti, and H. reticulatum are more closely related to Lycoris and Narcissus than to Allium and Agapanthus. Our results will provide information on the study of origins or relatedness of native species, and the identification of cultivars.

The Phytochemistry and Pharmacology of Tulbaghia, Allium, Crinum and Cyrtanthus: 'Talented' Taxa from the Amaryllidaceae.

Amaryllidaceae is a significant source of bioactive phytochemicals with a strong propensity to develop new drugs. The genera Allium, Tulbaghia, Cyrtanthus and Crinum biosynthesize novel alkaloids and other phytochemicals with traditional and pharmacological uses. Amaryllidaceae biomolecules exhibit multiple pharmacological activities such as antioxidant, antimicrobial, and immunomodulatory effects. Traditionally, natural products from Amaryllidaceae are utilized to treat non-communicable and infectious human diseases. Galanthamine, a drug from this family, is clinically relevant in treating the neurocognitive disorder, Alzheimer's disease, which underscores the importance of the Amaryllidaceae alkaloids. Although Amaryllidaceae provide a plethora of biologically active compounds, there is tardiness in their development into clinically pliable medicines. Other genera, including Cyrtanthus and Tulbaghia, have received little attention as potential sources of promising drug candidates. Given the reciprocal relationship of the increasing burden of human diseases and limited availability of medicinal therapies, more rapid drug discovery and development are desirable. To expedite clinically relevant drug development, we present here evidence on bioactive compounds from the genera Allium, Tulgbaghia, Cyrtanthus and Crinum and describe their traditional and pharmacological applications.

Comparative Analysis of the Complete Chloroplast Genomes in Allium Section Bromatorrhiza Species (Amaryllidaceae): Phylogenetic Relationship and Adaptive Evolution.

With the development of molecular sequencing approaches, many taxonomic and phylogenetic problems of the genus Allium L. have been solved; however, the phylogenetic relationships of some subgenera or sections, such as section Bromatorrhiza, remain unresolved, which has greatly impeded our full understanding of the species relationships among the major clades of Allium. In this study, the complete chloroplast (cp) genomes of nine species in the Allium sect. Bromatorrhiza were determined using the Illumina paired-end sequencing, the NOVOPlasty de novo assembly strategy, and the PGA annotation method. The results showed that the cp genome exhibited high conservation and revealed a typical circular tetrad structure. Among the sect. Bromatorrhiza species, the gene content, SSRs, codon usage, and RNA editing site were similar. The genome structure and IR regions' fluctuation were investigated while genes, CDSs, and non-coding regions were extracted for phylogeny reconstruction. Evolutionary rates (Ka/Ks values) were calculated, and positive selection analysis was further performed using the branch-site model. Five hypervariable regions were identified as candidate molecular markers for species authentication. A clear relationship among the sect. Bromatorrhiza species were detected based on concatenated genes and CDSs, respectively, which suggested that sect. Bromatorrhiza is monophyly. In addition, there were three genes with higher Ka/Ks values (rps2, ycf1, and ycf2), and four genes (rpoC2, atpF, atpI, and rpl14) were further revealed to own positive selected sites. These results provide new insights into the plastome component, phylogeny, and evolution of Allium species.

Biotechnological Approaches to Optimize the Production of Amaryllidaceae Alkaloids.

Amaryllidaceae alkaloids (AAs) are plant specialized metabolites with therapeutic properties exclusively produced by the Amaryllidaceae plant family. The two most studied representatives of the family are galanthamine, an acetylcholinesterase inhibitor used as a treatment of Alzheimer's disease, and lycorine, displaying potent in vitro and in vivo cytotoxic and antiviral properties. Unfortunately, the variable level of AAs' production in planta restricts most of the pharmaceutical applications. Several biotechnological alternatives, such as in vitro culture or synthetic biology, are being developed to enhance the production and fulfil the increasing demand for these AAs plant-derived drugs. In this review, current biotechnological approaches to produce different types of bioactive AAs are discussed.

Non-invasive assessment of the physiological role of leaf aerenchyma in Hippeastrum Herb. and its relation to plant water status.

MAIN CONCLUSION: The leaf patch clamp pressure probe combined with gas exchange measurements provides a non-invasive approach for measuring leaf aerenchyma pressure and study its physiological role in plants. The non-invasive leaf patch clamp pressure probe (LPCP) measures the output pressure, Pp, in response to the pressure applied by two magnets clamped to a leaf. In many plant species, it has been observed that the diel pattern of Pp follows the changes in the leaf turgor pressure reversely. The genus Hippeastrum comprises 143 species and many hybrids and cultivars of high economic value within Amaryllidaceae. Their leaves are characterized by the presence of aerenchyma composed of lacunae, running throughout the leaf and composing most of the mesophyll volume. In Hippeastrum, the diel changes of the LPCP output pressure are the reverse of that observed on the air pressure in the leaf aerenchyma, Pa, which depends on the changes in the leaf vapor pressure occurring during photosynthesis. A theoretical model is proposed and confirmed experimentally by LPCP and gas exchange measurements. The output pressure, Pp, in Hippeastrum can be related to the plant water status through the gas exchange processes that occur during photosynthesis. Considering the natural habitats of Hippeastrum species, these results agree with the physiological role of leaf aerenchyma in facilitating gas transport and light scattering in leaves, thus contributing to the photosynthetic efficiency of these plants under adverse environments. A second, but supplemental, interpretation of the LPCP output pressure, Pp, when applied on species in which the aerenchyma constitutes most of the mesophyll volume is presented.

Lycorine and organ protection: Review of its potential effects and molecular mechanisms.

BACKGROUND: Multiorgan dysfunction, especially sepsis-related multiorgan damage, remains a major cause of high mortality in the late stages of infection and a great clinical challenge. In recent years, natural drugs have received widespread attention because of their low cost, wide sources, high efficacy, low toxicity, and limited side effects. Lycorine, a natural compound extracted from Amaryllidaceae, exhibits multiple pharmacological activities, including in the regulation of autophagy and the induction of cancer cell apoptosis, and has anti-inflammatory, antifungal, antiviral, antimalarial, and antitumor activities. However, studies on lycorine have mainly focused on its antitumor properties, and research on its use for organ protection, especially in sepsis-related organ injury, is relatively limited. PURPOSE: To review and discuss the effects and mechanisms of lycorine in the treatment of multi-organ dysfunction, especially sepsis. METHODS: Literature searches in electronic databases, such as Web of Science, Science Direct, PubMed, Google Scholar, and Scopus, were performed using 'Lycorine', 'Amaryllidaceae', 'Pharmacology', 'Pharmacokinetics', 'Anti-inflammation', 'Autophagy', 'Apoptosis', 'Anti-microbial and anti-parasitic', 'Antitumor', 'Organ protection', and 'Sepsis' as keywords, the correlated literature was extracted and conducted from the databases mentioned above. RESULTS: By summarizing the progress made in existing research, we found that the general effects of lycorine involve the regulation of autophagy and the induction of cancer cell apoptosis, and anti-inflammatory, antifungal, antiviral, antimalarial, and antitumor effects; through these pathways, the compound can ameliorate organ damage. In addition, lycorine was found to have an important effect on organ damage in sepsis. CONCLUSION: Lycorine is a promising natural organ protective agent. This review will provide a new theoretical basis for the treatment of organ protection, especially in sepsis.

Seed macro- and micromorphology in Allium (Amaryllidaceae) and its phylogenetic significance.

BACKGROUND AND AIMS: Macro- and micromorphology of seeds are diagnostic characteristics of importance in delimiting taxa in Allium (Amaryllidaceae). However, there is no consensus on the phylogenetic significance of testa cell characteristics and whether they reflect the different evolutionary levels recognized in Allium. METHODS: Seeds of 95 species (98 samples) representing 14 subgenera and 58 sections of Allium were examined using scanning electron microscopy (SEM) for such traits as periclinal wall surface area of ten testa cells, distance between testa cells (macromorphology), testa cell shapes, and arrangement and structure of anticlinal and periclinal walls (micromorphology). The data matrix was subjected to cladistic analysis. The produced phylogenetic tree was examined against the molecular tree obtained from publically available ITS sequences. KEY RESULTS: The periclinal wall surface area of ten testa cells and the distance between them, examined for the first time, were found useful for delimitation of species in Allium. Based on seed macro- and micromorphology, we present a taxonomic key and a hypothetical reconstruction of the migration routes during the early stages of evolution of Allium. CONCLUSIONS: The ancestors of Allium originated in an area bounded by the Caucasus, Central Asia and Iran. The seed testa morphology-based evolutionary state of a species is determined by two parameters: the shape of the periclinal walls and curvature of the anticlinal walls.

Hymenocallis littoralis ameliorates inflammatory responses in LPS-stimulated RAW264.7 cells and HCl/EtOH-induced gastric mucosal injury via targeting the MAPK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Hymenocallis littoralis (Jacq.) Salisb. Also known as Pancratium littorale Jacq. And Hymenocallis panamensis Lindl., is a medicinal plant from the family Amarylideceae used for emetic and wound healing and has manifested anti-neoplastic, anti-oxidant, and anti-viral properties. AIM OF THE STUDY: The aim of this paper is to investigate the anti-inflammatory potential and molecular mechanism of H. littoralis against lipopolysaccharide (LPS)-induced macrophages and in vivo HCl/EtOH-induced gastritis mucosal injury models. MATERIALS AND METHODS: The production of pro-inflammatory cytokines and mediators was evaluated by Griess assay, RT-PCR, and real-time PCR. Moreover, the relevant proteins of mitogen-activated protein kinases (MAPKs) including ERK, JNK, p38, c-Jun, and c-Fos were detected using immunoblotting. RESULTS: We demonstrated that H. littoralis prominently dampened production of nitric oxide (NO) in LPS-, poly I:C-, or pam3CSK-stimulated RAW264.7 cells; down-regulated the expression levels of interleukin 6 (IL-6) and inducible nitric oxide synthase; and markedly attenuated the luciferase activities of AP-1 reporter promoters. Moreover, H. littoralis administration prominently downregulated c-Fos and c-Jun phosphorylation as well as JNK1, ERK2, and MKK7 overexpression in HEK 293T cells. Furthermore, H. littoralis displayed anti-inflammatory effects in the HCl/EtOH-induced gastritis mice model. CONCLUSIONS: Cumulatively, these results demonstrated that H. littoralis exerts eminently anti-inflammatory activities in LPS-stimulated RAW264.7 cells in vitro and in HCl/EtOH-induced gastritis mice models in vivo. These activities could be attributed to its modulatory effects on the MAPK signaling pathway.

Lycorine Alkaloid and Crinum americanum L. (Amaryllidaceae) Extracts Display Antifungal Activity on Clinically Relevant Candida Species.

Candida species are the main fungal agents causing infectious conditions in hospital patients. The development of new drugs with antifungal potential, increased efficacy, and reduced toxicity is essential to face the challenge of fungal resistance to standard treatments. The aim of this study is to evaluate the in vitro antifungal effects of two crude extracts of Crinum americanum L., a rich alkaloid fraction and lycorine alkaloid, on the Candida species. As such, we used a disk diffusion susceptibility test, determined the minimum inhibitory concentration (MIC), and characterized the components of the extracts using Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ESI FT-ICR MS). The extracts were found to have antifungal activity against various Candida species. The chemical characterization of the extracts indicated the presence of alkaloids such as lycorine and crinine. The Amaryllidaceae family has a promising antifungal potential. Furthermore, it was found that the alkaloid lycorine directly contributes to the effects that were observed for the extracts and fraction of C. americanum.

Complete genome organization and characterization of Hippeastrum latent virus.

The complete genome sequences of two carlaviruses were determined by high-throughput sequencing of RNA extracted from ringspot and mosaic, disease symptoms on leaves of spider lily plants (Crinum asiaticum, family Amaryllidaceae) growing as landscape plants in Hawaii. One, named Nerine latent virus (NeLV)-Hawaii with a genome of 8281 nucleotide exhibited the highest nucleotide identity and amino acid similarity of 95.5% and 96.0%, respectively, to the genome sequence of an isolate of NeLV from Narcissus sp. in Australia (JQ395044). The second, named Hippeastrum latent virus (HiLV)-Hawaii with a genome of 8497 nucleotides exhibited the highest nucleotide identity and amino acid similarity, 84.3% and 88.7%, respectively, to the sequence of a previously uncharacterized HiLV isolate from a potted flowering plant, Amaryllis (Hippeastrum hybridum Hort) in Taiwan (DQ098905). The amino acid sequence similarities of replicase (Rep) and coat protein (CP) between HiLV-Hawaii and NeLV-Hawaii were 44.8% and 38.4%, respectively. Results of viral protein Rep and CP amino acid sequence comparisons from various carlaviruses provide evidence that HiLV and NeLV, previously classified as synonymous viruses are in fact unique viruses. This is the first report for the complete sequence, organization, and phylogenetic characterization of HiLV and the first detection of HiLV both in C. asiaticum and in the USA.

Complete genome sequence of Amazon lily mosaic virus isolated from amaryllis (Hippeastrum hybridum Hort.).

In April 2011, a virus was isolated by single-lesion isolation on Chenopodium quinoa leaves from an amaryllis plant with chlorotic ringspots in a private garden in Changhua County, Taiwan. An Illumina MiSeq sequencing system was used to determine the genomic nucleotide (nt) sequence of the virus. A de novo-assembled contig with 9377 nt, containing an open reading frame encoding a putative potyviral polyprotein, was annotated as the potyvirus Amazon lily mosaic virus (ALiMV), sharing 95.5% nt sequence identity with a partial genomic sequence of ALiMV available in the GenBank database. Therefore, the amaryllis virus was designated as ALiMV-TW. Through 5´ and 3´ rapid amplification of cDNA ends (RACE), the complete 9618-nt genome sequence of ALiMV-TW was determined. Sequence comparisons indicated that the genome and polyprotein of ALiMV-TW share 52.3-65.1% nt and 30.1-64.2% aa sequence identity, respectively, with those of other potyviruses. This is the first report of a complete genome sequence of ALiMV.

In vitro and in silico analysis of galanthine from Zephyranthes carinata as an inhibitor of acetylcholinesterase.

Zephyranthes carinata Herb., a specie of the Amaryllidoideae subfamily, has been reported to have inhibitory activity against acetylcholinesterase. However, scientific evidence related to their bioactive alkaloids has been lacking. Thus, this study describes the isolation of the alkaloids of this plant, and their inhibition of the enzymes acetylcholinesterase (eeAChE) and butyrylcholinesterase (eqBuChE), being galanthine the main component. Additionally, haemanthamine, hamayne, lycoramine, lycorine, tazettine, trisphaeridine and vittatine/crinine were also isolated. The results showed that galanthine has significant activity at low micromolar concentrations for eeAChE (IC50 = 1.96 µg/mL). The in-silico study allowed to establish at a molecular level the high affinity and the way galanthine interacts with the active site of the TcAChE enzyme, information that corroborates the result of the experimental IC50. However, according to molecular dynamics (MD) analysis, it is also suggested that galanthine presents a different inhibition mode that the one observed for galanthamine, by presenting interaction with peripheral anionic binding site of the enzyme, which prevents the entrance and exit of molecules from the active site. Thus, in vitro screening assays plus rapid computer development play an essential role in the search for new cholinesterase inhibitors by identifying unknown bio-interactions between bioactive compounds and biological targets.

Anti-Trypanosoma cruzi activity of alkaloids isolated from Habranthus brachyandrus (Amaryllidaceae) from Argentina.

BACKGROUND: Chagas disease, caused by the parasite Trypanosoma cruzi, affects over six million people worldwide, mainly in Latin American countries. Currently available drugs have variable efficacy in the chronic phase and significant side effects, so there is an urgent need for safer chemotherapeutic treatments. Natural products provide privileged structures that could serve as templates for the synthesis of new drugs. Among them, Amaryllidaceae plants have proved to be a potential natural source of therapeutical agents due to their rich diversity in alkaloids. PURPOSE: To identify alkaloids with anti-T. cruzi activity from Habranthus brachyandrus (Baker) Sealy (Amaryllidaceae, subfamily Amaryllidoideae) collected in Argentina. METHODS: An H. brachyandrus alkaloid extract was tested against T. cruzi, and its cytotoxicity profile was evaluated against two mammalian cell lines to ascertain its selectivity against the parasite and potential liver toxicity. It was also assessed by a stage-specific anti-amastigote assay and analysed by GC/MS to determine its alkaloid profile. The isolated alkaloids were also tested using the aforementioned assays. RESULTS: The extract showed high and specific activity against T. cruzi. The alkaloids lycoramine, galanthindole, 8-O-demethylmaritidine, 8-O-demethylhomolycorine, nerinine, trisphaeridine, deoxytazettine, and tazettamide were identified by means of GC-MS. In addition, hippeastidine (also named aulicine), tazzetine, ismine, and 3-epimacronine were isolated. The alkaloid ismine was specifically active against the parasite and had low toxicity against HepG2 cells, but did not show anti-amastigote activity. CONCLUSION: The extract had specific anti-T. cruzi activity and the isolated alkaloid ismine was partially responsible of it. These results encourage further exploration of H. brachyandrus alkaloids in search of novel starting points for Chagas disease drug development.