Insights into Neogene Mediterranean biogeography based on phylogenetic relationships of mountain and lowland lineages of Narcissus (Amaryllidaceae)

Aim Our aims were: (1) to reconstruct the phylogenetic relationships of daffodils (Narcissus), focusing on the lowland subgenus Hermione and the mountain section Apodanthi; (2) to estimate the temporal setting of diversification; (3) to reconstruct the migration patterns of the lineages; and (4) to examine the microevolutionary differentiation of the wide‐ranging Narcissus tazetta group across the Mediterranean. Location The Mediterranean Basin. Methods Plastid (trnT–L, trnL–F and ndhF) sequences were obtained from 63 populations representing 23 species of Narcissus and combined with published data from 16 species. Phylogenetic relationships and dating were inferred by Bayesian analysis based on geological events and divergence estimates of closely related taxa. A dispersal–extinction–cladogenesis analysis was performed using maximum likelihood methods to infer ancestral geographical distributions, and phylogeographical reconstruction was performed using coalescence analysis. Results Subgenus Hermione is not recognized as a monophyletic group because two of the nine species were found to have a close relationship with the subgenus Narcissus. The results on section Apodanthi confirmed previous findings of its monophyly and phylogenetic relationships within this mountain group. Molecular dating and ancestral range reconstructions suggest that the ancestor of Narcissus originated in the Iberian Peninsula during the Late Oligocene–Early Miocene. Eastward expansion of the lineage range proceeded from the western Mediterranean and involved colonization of mountain ranges in northern Africa. The phylogeography of the N. tazetta group revealed a widespread distribution of certain haplotypes, suggesting wide dispersal and a high level of colonization in the Mediterranean Basin. Main conclusions Our study points to the role of three key historical events in Narcissus diversification: tectonic shifts of the Alboran domain in the western Mediterranean, the Messinian salinity crisis, and the onset of the Mediterranean climate followed by periods of repeated glaciation. Diversification of section Apodanthi probably resulted from allopatric speciation, while subgenus Hermione may have shown more sympatric speciation and high dispersal, despite the lack of apparent adaptations to long‐distance dispersal. This is best exemplified by the presence of both ancestral and recent haplotypes of N. tazetta across the Mediterranean.     

The development of eight microsatellite loci in the wild daffodil Narcissus triandrus (Amaryllidaceae)

We report microsatellite primer pairs for the wild tristylous daffodil, Narcissus triandrus (Amaryllidaceae). From enriched libraries, we identified 58 unique microsatellite loci. We designed primer pairs for 27 of these loci and screened genomic DNA from 38 to 40 adults from a single population. For eight polymorphic loci, the number of alleles per locus ranged from five to 17. As six primers also amplified loci in three other Narcissus species, including two horticultural varieties, we expect that some of these markers will be transferable to other Narcissus species.     

Clinanthus microstephium,an Amaryllidaceae Species with Cholinesterase Inhibitor Alkaloids: Structure−Activity Analysis of Haemanthamine Skeleton Derivatives

Plants of the Amaryllidaceae family are well‐known (not only) for their ornamental value but also for the alkaloids that they produce. In this report, the first phytochemical study of Clinanthus genus was carried out. The chemical composition of alkaloid fractions from Clinanthus microstephium was analyzed by GC/MS and NMR. Seven known compounds belonging to three structural types of Amaryllidaceae alkaloids were identified. An epimeric mixture of a haemanthamine‐type compound (6‐hydroxymaritidine) was tested as an inhibitor against acetyl‐ and butyrylcholinesterase enzymes (AChE and BChE, respectively), two enzymes relevant in the treatment of Alzheimer's disease, with good results. Structure–activity relationships through molecular docking studies with this alkaloid and other structurally related compounds were discussed.     

Evidence of a novel allergenic protein Narcin in the bulbs of Narcissus tazetta

Several plant-derived allergens have been identified which result in the formation of immunoglobulin E antibodies. Primarily, these allergens belong to the protein families including seed storage proteins, structural proteins and pathogenesis-related proteins. Several allergens are also reported from flower bulbs which cause contact dermatitis. Such symptoms are highly common with the bulb growers handling different species of Narcissus. Narcissus toxicity is also reported if the bulbs are consumed accidentally. The present study aimed to characterize the protein from the bulbs of Narcissus tazetta responsible for its allergenic response. A 13 kDa novel allergenic protein, Narcin was isolated from the bulbs of Narcissus tazetta. The protein was extracted using ammonium sulfate fractionation. The protein was further purified by anion exchange chromatography followed by gel filtration chromatography. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation. The allergenicity of the protein was measured by cytokine production using flow cytometry in peripheral blood mononuclear cells. Further estimation of total IgE was performed by ELISA method. This novel protein was found to induce pro-inflammatory cytokines and thus induce allergy by elevating total IgE level. The novel protein, Narcin isolated from Narcissus tazetta was found to exhibit allergenic properties.     

Fungal endophytes promote the accumulation of Amaryllidaceae alkaloids in Lycoris radiata

Lycoris radiata is a main source of Amaryllidaceae alkaloids; however, the low content of these alkaloids in planta remains a limit to their pharmaceutical development and utilization. The accumulation of secondary metabolites can be enhanced in plants inoculated with fungal endophytes. In this study, we analysed the diversity of culturable fungal endophytes in different organs of L. radiata. Then, by analysing the correlation between the detectable rate of each fungal species and the content of each tested alkaloid, we proposed several fungal candidates implicated in the increase of alkaloid accumulation. This was verified by inoculating these candidates to L. radiata plants. Based on the results of two independent experiments conducted in May 2018 and October 2019, the individual inoculation of nine fungal endophytes significantly increased the total content of the tested alkaloids in the entire L. radiata plants. This is the first study in L. radiata to show that fungal endophytes are able to improve the accumulation of various alkaloids. Therefore, our results provide insights into a better understanding of interactions between plants and fungal endophytes and suggest an effective strategy for enhancing the alkaloid content in the cultivation of L. radiata.     

Plants of the ‘Libellus de Medicinalibus Indorum Herbis’ from Mexico, 1552. Zephyranthes fosteri (Amaryllidaceae) Alkaloids

The Libellus de Medicinalibus Indorum Herbis (Booklet of Indian Medicinal Plants) is the first book of medicinal plants written in the American continent. It was first published in 1939 as ‘An Aztec Herbal’. One of the depicted plants is Huetzcanixochitl (laughing flower) interpreted as Zephyranthes fosteri (Amaryllidaceae). No chemical or pharmacological studies are reported for this species; so, we decide to investigate it. The GC/MS of the bulbs and aerial parts extracts indicated that they contain Amaryllidaceae alkaloids, among them: lycorine, 3-O-acetylpowelline, and norlycoramine. An unknown major alkaloid was isolated and identified by ¹H, ¹³C-NMR and MS, as 3′-demethoxy-6-epimesembranol ( 1 ). The methanolic extract, the alkaloid fraction, and compound 1 inhibited acetylcholinesterase in vitro. Mesembrine alkaloids are found in Sceletium species (Aizoaceae). Several are known as serotonin recapture inhibitors and have been proposed as potential antidepressant drugs. The presence of 1 suggests that Z. fosteri was probably used in pre-Columbian times in Mexico as a ‘stimulant and euphoriant’, alike Sceletium tortuosum by several ethnic groups in South Africa.     

Structural Characterisation of Alkaloids in Leaves and Roots of Stephania kwangsiensis by LC‐QTOF‐MS

Introduction

The tuberous roots of Stephania kwangsiensis, which contain bioactive alkaloids, are used as a traditional Chinese medicine. Overexploitation of the roots has made the plant increasingly rare, and the abundant leaves of the same plant may offer a potential alternative. However, there is insufficient phytochemical information for a comparison of alkaloid compositions in the two parts.

Objective

To characterise and compare the alkaloids in the leaves and roots of S. kwangsiensis.

Methods

The alkaloids in S. kwangsiensis were characterised using high pressure liquid chromatography coupled with positive electrospray ionisation quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐(+)ESI‐QTOF‐MS/MS). The alkaloid compositions in the leaves and roots were compared by visual inspection combined with principal component analysis (PCA) of the HPLC‐MS data.

Results

Seventy‐five alkaloids comprising aporphine‐, proaporphine‐, protoberberine‐, benzylisoquinoline‐, bisbenzylisoquinoline‐ and morphine‐type alkaloids were identified or tentatively identified in the roots and leaves of S. kwangsiensis. Sixty‐three of these alkaloids have not been previously reported in this species, and three have not been previously reported in the literature. The roots and leaves had similarities in alkaloid composition but differences in the peak intensities of most alkaloids. The PCA revealed that the samples were clustered into two distinct groups, which corresponded to leaves and roots.

Conclusion

This study further clarified the chemical constituents in the roots of S. kwangsiensis, and revealed that diverse alkaloids were also present in the leaves. The comparative chemical profiling of the two parts provides useful information on their potential medicinal use. Copyright © 2017 John Wiley & Sons, Ltd.     

Influence of plant origin on propagation capacity and alkaloid biosynthesis during long-term <Emphasis Type="Italic">in vitro</Emphasis> cultivation of <Emphasis Type="Italic">Leucojum aestivum</Emphasis> 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.     

Callus induction, somatic embryogenesis and organogenesis in Narcissus confusus: correlation between the state of differentiation and the content of galanthamine and related alkaloids

In vitro cultures from two strains of Narcissus confusus (Amaryllidaceae) initiated from mature seeds were screened for their ability to produce alkaloids. Protocols for callus induction, somatic embryogenesis and organogenesis were established. The alkaloid contents were determined by HPLC. Undifferentiated calli produced small amounts of galanthamine, which increased with the degree of tissue differentiation. Scanning electron micrographs of the cultures at different stages of development were made. Embryogenic friable calli were maintained in culture for more than 2 years, retaining a high regeneration capability. All regenerated plants showed normal morphological characteristics. Received: 20 August 1997 / Revision received: 20 December 1997 / Accepted: 1 February 1998     

Antiprotozoal alkaloids from Galanthus trojanus

Two new alkaloid N-oxides, 1-O-acetyldihydromethylpseudolycorine N-oxide, and 11-hydroxyvittatine N-oxide, ten known alkaloids; arolycoricidine, haemanthamine, O-methylnorbelladine, narcidine, dihydrolycorine, 8-O-demethylmaritidine, stylopine and protopine, nicotinic acid and tyramine were isolated from Galanthus trojanus A.P. Davis & N. Özhatay (Amaryllidaceae). The chemical structures of the isolates were elucidated by UV, IR, MS, CD, 1D and 2D NMR experiments. The in vitro antiprotozoal and cytotoxic potentials of the compounds were also evaluated.     

Antiprotozoal alkaloid principles of the plant family Amaryllidaceae

Protozoan-borne diseases are prominent amongst diseases caused by parasites. Given their alarming morbidity and mortality statistics, there is ever growing interest in new therapies against these diseases. Whilst synthetic drugs such as benznidazole and melarsoprol have had a profound influence on the clinical setup, there has been significant interest in the phytochemical platform to also deliver such drug candidates. The plant family Amaryllidaceae is recognizable for its isoquinoline alkaloids, which exhibit attractive molecular architectures and interesting biological properties. This survey focuses on the antiprotozoal activities of 73 of such substances described in 18 different species of the Amaryllidaceae. Of these, 2-O-acetyllycorine was identified as the most potent (IC₅₀ 0.15 μg/mL against Trypansoma brucei brucei). Also considered are structure-activity relationships which have served to modulate activities, as well as the plausible mechanisms that underpin these effects and afford insight to the Amaryllidaceae alkaloid antiprotozoal pharmacophore.     

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

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

Biogeographical Patterns and Phenological Changes in Lapiedra martinezii Lag. Related to Its Alkaloid Diversity

The aim of this work was to investigate the alkaloid patterns of Lapiedra martinezii and their relation to biogeography and phenology focused in a phylogenetic comparison. Plants from 14 populations of L. martinezii, covering almost its entire distribution area, were subjected to morphological, ecological, and phytochemical analysis. Experiments for different alkaloid‐type content are proposed as a new tool for analysis of plant distribution. Several plants were transplanted for weekly observation of their phenological changes, and alkaloids from different plant organs were extracted, listed, and compared. The alkaloid pattern of L. martinezii comprises 49 compounds of homolycorine, lycorine, tazettine, haemantamine, and narciclasine types. The populations located in the north and south margins of the distribution area displayed alkaloid patterns different from those of the central area. Changes in these patterns during their phenological cycle may be related to a better defence for plant reproduction. L. martinezii is an old relict plant, and it has maintained some of the more primitive morphological features and alkaloid profiles of the Mediterranean Amaryllidaceae. The variations in alkaloid content observed could be interpreted in a phylogenetic sense, and those found in their phenological changes, in an adaptive one.     

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.     

The Alkaloids of Fungi

Examination of different species of local fungi, grown on two nutritive solutions of different composition, for alkaloid formation was investigated. The formation of alkaloids was confined to four species, namely: Geotrichum candidum, Mucor hiemalis, Aspergillus flavus and Rhizopus nigricans. A comparative study of the growth as well as the formation of alkaloids by these species and by Claviceps purpurea NRRL was carried out. Methods were also described with which the different alkaloids produced by the experimental strains were identified. Peptides as well as clavine type alkaloids were detected in all cases except with Mucor hiemalis where a compound corresponding to ergosine was the only alkaloid present.     

New skeletons and new concepts in Amaryllidaceae alkaloids

The Amaryllidaceae alkaloids, represent a group of isoquinoline alkaloids, which are produced almost solely by members of the Amaryllidaceae family. The alkaloids of this family have attracted considerable amount of interest due to some important pharmacological activities they were shown to possess. In the last decade, our phytochemical studies on four Galanthus (Amaryllidaceae) species of Turkish origin have yielded quite a number of new alkaloids with diverse structures. Among these alkaloids, gracilines and plicamines constitute two new subgroups for the Amaryllidaceae alkaloids. The gracilines contain an incorporated 10b,4a-ethanoiminodibenzo[b,d]pyrane skeleton. The plicamines are dinitrogenous compounds, where the oxygen atom in position 7 of a tazettine skeleton is replaced by a nitrogen atom substituted by a pendant 4-hydroxyphenethyl moiety. One of the new alkaloids, galanthindole, which possesses a nonfused indole ring, unlike the already known subgroups of Amaryllidaceae alkaloids, may be considered as the prototype of a third new subgroup of the Amaryllidaceae alkaloids. Additionally, two known isoquinoline alkaloids which do not possess one of the established skeletons of the Amaryllidaceae alkaloids, namely ( − )-capnoidine and (+)-bulbocapnine, have been isolated from a Turkish Galanthus species. Totally, 21 new, 20 known alkaloids and 2 known lignans have been characterized. In this review, the isolation and structure elucidation of these compounds with interesting chemical structures are described.     

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

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

Quantification of Anti‐Addictive Alkaloids Ibogaine and Voacangine in In Vivo‐ and In Vitro‐Grown Plants of Two Mexican Tabernaemontana Species

Tabernaemontana alba and Tabernaemontana arborea are Apocynaceae species used in Mexican traditional medicine for which little phytochemical information exists. In this study, preliminary gas chromatography/mass spectrometry analyses of different organs obtained from wild plants of both species identified a total of 10 monoterpenoid indole alkaloids (MIAs) and one simple indole alkaloid, nine of which were reported for the first time in these species. Furthermore, callus cultures were established from T. alba leaf explants and regeneration of whole plants was accomplished via somatic embryogenesis. The anti‐addictive MIAs ibogaine and voacangine were then quantified by gas chromatography with flame ionization detection in wild plants of both species, as well as greenhouse‐grown plants, in vitro‐grown plantlets and embryogenic callus of T. alba. Ibogaine and voacangine were present in most samples taken from the whole plants of both species, with stem and root barks showing the highest concentrations. No alkaloids were detected in callus samples. It was concluded that T. alba and T. arborea are potentially viable sources of ibogaine and voacangine, and that these MIAs can be produced through somatic embryogenesis and whole plant regeneration of T. alba. Approaches to increase MIA yields in whole plants and to achieve alkaloid production directly in cell cultures are discussed.     

Morphological and chemical variation of Stemona tuberosa from southern China – Evidence for heterogeneity of this medicinal plant species

• The occurrence of bioactive alkaloids and tocopherols was studied in 15 different provenances of Stemona tuberosa Lour. collected in southern China, to examine chemical variation of individuals that show notable differences in flower characteristics. Morphological variations stimulated examination of chemical characteristics of these individuals.
• Methanolic root extracts of 15 individuals of S. tuberosa were comparatively assessed with HPLC‐UV‐DAD/ELSD. Five of seven compounds were co‐chromatographically identified. Two compounds were isolated and their structure elucidated using NMR and MS. Amounts of alkaloids and tocopherols were determined using HPLC‐UV‐DAD/ELSD with the external standard method.
• Five alkaloids, tuberostemonine ( 1 ), tuberostemonine A ( 2 ), neotuberostemonine ( 3 ), tuberostemonine N ( 4 ), stemoninine ( 5 ) and two 3,4‐dehydrotocopherol derivatives were identified. Within S. tuberosa alkaloid accumulation tends either towards tuberostemonine ( 1 ) or stemoninine ( 5 ). All individuals show a notable co‐occurrence of compounds 1 or 5 and 3,4‐dehydro‐δ‐tocopherol ( 6 ). These results coincide with differences in flower morphology of S. tuberosa.
• Stemona tuberosa, as defined in the Flora of China, shows a remarkable variation in flower morphology and additionally in the accumulation of alkaloids. The obtained data show the need for future species delimitation to either species or subspecies level.

     

Alkaloids of Datura ceratocaula

Thirty-six alkaloids were identified in the organs of Datura ceratocaula by GC/MS. Thirty-three of them have not been previously reported for the species. Furthermore, a new tropane ester was tentatively identified as 3-(3'-formyloxytropoyloxy)tropane on basis of its mass spectral fragmentation. Hyoscyamine was the main alkaloid in the plant organs.