Unveiling Amaryllidaceae alkaloids: From biosynthesis to antiviral potential - a review

Abstract Covering: 2017 to 2023 (now) Amaryllidaceae alkaloids (AAs) are a unique class of specialized metabolites containing heterocyclic nitrogen bridging that play a distinct role in higher plants. Irrespective of their diverse structures, most AAs are biosynthesized via intramolecular oxidative coupling. The complex organization of biosynthetic pathways is constantly enlightened by new insights owing to the advancement of natural product chemistry, synthetic organic chemistry, biochemistry, systems and synthetic biology tools and applications. These promote novel compound identification, trace-level metabolite quantification, synthesis, and characterization of enzymes engaged in AA catalysis, enabling the recognition of biosynthetic pathways. A complete understanding of the pathway benefits biotechnological applications in the long run. This review emphasizes the structural diversity of the AA specialized metabolites involved in biogenesis although the process is not entirely defined yet. Moreover, this work underscores the pivotal role of synthetic and enantioselective studies in justifying biosynthetic conclusions. Their prospective candidacy as lead constituents for antiviral drug discovery has also been established. However, a complete understanding of the pathway requires further interdisciplinary efforts in which antiviral studies address the structure-activity relationship. This review presents current knowledge on the topic

Rooted in therapeutics: Comprehensive analyses of Cannabis sativa root extracts reveals potent antioxidant, anti-inflammatory, and bactericidal properties

Abstract Following the legalization of recreational Cannabis in Canada in 2018, the associated waste, including Cannabis roots, has significantly increased. Cannabis roots, comprising 30%–50% of the total plant, are often discarded despite their historical use in Ayurvedic medicine for treating inflammatory and infectious disorders. This study evaluates the phytochemical and therapeutic properties of Cannabis root extracts from a high tetrahydrocannabinolic acid, low cannabidiolic acid cultivar (variety Alien Gorilla Glue). We performed ultra high-performance liquid chromatography coupled with mass spectrometry (UPLC-QTOF-MS) to identify the chemical components of the Cannabis roots. Extracts using water, ethanol and acid-base solvents were tested for antioxidant activity through free radical scavenging, metal chelation, and lipoperoxidation inhibition assays. Mitochondrial membrane protection was assessed using flow cytometry with the MitoPerOx probe in THP-1 monocytic leukemia cells. Anti-inflammatory potential was evaluated by measuring interleukin-6 levels in lipopolysaccharide-stimulated THP-1 cells. Bactericidal/fungicidal efficacy against Escherichia coli, Staphylococcus aureus, and Candida albicans was determined using the p-iodonitrophenyltetrazolium assay. Additionally, we investigated the anticholinesterase activity of Cannabis root extracts, given the potential role of plant alkaloids in inhibiting cholinesterase, an enzyme targeted in Alzheimer’s disease treatments. UPLC-QTOF-MS analysis suggested the presence of several phenolic compounds, cannabinoids, terpenoids, amino acids, and nitrogen-containing compounds. Our results indicated significant antioxidant, bactericidal, and anticholinesterase properties of Cannabis root extracts from both soil and hydroponic cultivation. Extracts showed strong antioxidant activity across multiple assays, protected mitochondrial membrane in THP-1 cells, and exhibited anti-inflammatory and bactericidal/fungicidal efficacy. Notably, soil-cultivated roots displayed superior anti-inflammatory effects. These findings demonstrate the remarkable antioxidant, anti-inflammatory, and anti-microbial activities of Cannabis roots, supporting their traditional uses and challenging their perception as mere waste. This study highlights the therapeutic potential of Cannabis roots extracts and suggests avenues for further research and application

Cytotoxicity and Antiviral Properties of Alkaloids Isolated from Pancratium maritimum

Ten Amaryllidaceae alkaloids (AAs) were isolated for the first time from Pancratium maritimum collected in Calabria region, Italy. They belong to different subgroups of this family and were identified as lycorine, which is the main alkaloid, 9-O-demethyllycorine, haemanthidine, haemanthamine, 11-hydroxyvittatine, homolycorine, pancracine, obliquine, tazettine and vittatine. Haemanthidine was isolated as a scalar mixture of two 6-epimers, as already known also for other 6-hydroxycrinine alkaloids, but for the first time they were separated as 6,11-O,O′-di-p-bromobenzoyl esters. The evaluation of the cytotoxic and antiviral potentials of all isolated compounds was undertaken. Lycorine and haemanthidine showed cytotoxic activity on Hacat cells and A431 and AGS cancer cells while, pancracine exhibited selective cytotoxicity against A431 cells. We uncovered that in addition to lycorine and haemanthidine, haemanthamine and pancracine also possess antiretroviral abilities, inhibiting pseudotyped human immunodeficiency virus (HIV)−1 with EC50 of 25.3 μM and 18.5 μM respectively. Strikingly, all the AAs isolated from P. maritimum were able to impede dengue virus (DENV) replication (EC50 ranged from 0.34–73.59 μM) at low to non-cytotoxic concentrations (CC50 ranged from 6.25 μM to >100 μM). Haemanthamine (EC50 = 337 nM), pancracine (EC50 = 357 nM) and haemanthidine (EC50 = 476 nM) were the most potent anti-DENV inhibitors. Thus, this study uncovered new antiviral properties of P. maritimum isolated alkaloids, a significant finding that could lead to the development of new therapeutic strategies to fight viral infectious diseases

Marine specialized metabolites: Unveiling Nature's chemical treasures from the deep blue

Abstract Marine specialized metabolites (MSM) represent a fascinating realm of chemical diversity with multifaceted functions across the spectrum of life on Earth. These metabolites serve as weapons, metal transporters, regulatory agents, and more. The conservation of genes responsible for their production over extensive evolutionary timescales underscores their selective advantage. Recent decades have witnessed an upsurge in MSM studies, driven by advancements in analytical techniques and the ever-growing accessibility of the aquatic environment. Marine macro and microorganisms offer a rich tapestry of specialized metabolites, some exhibiting potent activities in diverse domains, including medicine. The study of MSM presents several challenges, reflecting the need to separate complex mixtures into individual bioactive metabolites and utilize state-of-the-art extraction methods. Comprehensive structural analysis relies on advanced spectroscopic approaches, including nuclear magnetic resonance and mass spectrometry. These tools are instrumental in unravelling the chemical diversity of MSM and understanding their potential applications. While bioprospecting offers enormous potential, it raises critical challenges concerning sustainability, conservation, and equitable benefit-sharing. International protocols like the Nagoya Protocol seeks to regulate access to and share benefits from genetic resources, with considerable implications for marine bioprospecting. The convergence of advanced metabolomics, metagenomics, and synthetic biology offers promising avenues for accelerating the discovery and sustainable production of MSM, shaping the future of this field. This comprehensive review provides a deep dive into the challenges, methodologies, and emerging trends in studying marine-derived natural products, underscoring the immense potential of MSM for advancing chemical sciences and their transformative applications in diverse areas such as food, medicine, biotechnology, and environmental conservation. By bridging multiple disciplines, the continued exploration and sustainable utilization of these metabolites hold the promise of unlocking new innovations for society's benefit

Isolation and biological characterization of homoisoflavanoids and the alkylamide n‐p‐coumaroyltyramine from crinum biflorum rottb., an amaryllidaceae species collected in Senegal

Crinum biflorum Rottb. (syn. Crinum distichum) is an Amaryllidaceae plant used in African traditional medicine but very few studies have been performed on this species from a chemical and applicative point of view. Bulbs of C. biflorum, collected in Senegal, were extracted with ethanol by Soxhlet and the corresponding organic extract was purified using chromatographic methods. The pure compounds were chemically characterized by spectroscopic techniques (1D and 2D1H and13C NMR, HR MS and ECD) and X‐ray analysis. Four homoisoflavonoids (1–4) and one alkylamide (5) were isolated and characterized as 5,6,7‐trimethoxy‐3‐(4‐hydroxybenzyl)chroman‐4‐one (1), as 3‐hydroxy‐ 5,6,7‐trimethoxy‐3‐(4‐hydroxybenzyl)chroman‐4‐one (2), as 3‐hydroxy‐5,6,7‐trimethoxy‐3‐(4‐methox-ybenzyl)chroman‐4‐one (3) and as 5,6,7‐trimethoxy‐3‐(4‐methoxybenzyl)chroman‐4‐one (4), and the alkylamide as (E)‐N‐(4‐hydroxyphenethyl)‐3‐(4‐hydroxyphenyl)acrylamide (5), commonly named N‐ p‐coumaroyltyramine. The relative configuration of compound 1 was verified thanks to the X‐ray analysis which also allowed us to confirm its racemic nature. The absolute configurations of compounds 2 and 3 were assigned by comparing their ECD spectra with those previously reported for urgineanins A and B. Flavanoids 1, 3 and 4 showed promising anticancer properties being cytotoxic at low micromolar concentrations towards HeLa and A431 human cancer cell lines. The N‐p‐coumaroyltyramine (5) was selectively toxic to A431 and HeLa cancer cells while it protected immortalized HaCaT cells against oxidative stress induced by hydrogen peroxide. Compounds 1–4 also inhibited acetylcho-linesterase activity with compound 3 being the most potent. The anti‐amylase and the strong anti-glucosidase activity of compound 5 were confirmed. Our results show that C. biflorum produces compounds of therapeutic interest with anti‐diabetic, anti‐tumoral and anti‐acetylcholinesterase properties

Generation of Potent and Stable Human CD4+ T Regulatory Cells by Activation-independent Expression of FOXP3

Therapies based on enhancing the numbers and/or function of T regulatory cells (Tregs) represent one of the most promising approaches to restoring tolerance in many immune-mediated diseases. Several groups have investigated whether human Tregs suitable for cellular therapy can be obtained by in vitro expansion, in vitro conversion of conventional T cells into Tregs, or gene transfer of the FOXP3 transcription factor. To date, however, none of these approaches has resulted in a homogeneous and stable population of cells that is as potently suppressive as ex vivo Tregs. We developed a lentivirus-based strategy to ectopically express high levels of FOXP3 that do not fluctuate with the state of T-cell activation. This method consistently results in the development of suppressive cells that are as potent as Tregs and can be propagated as a homogeneous population. Moreover, using this system, both naive and memory CD4+ T cells can be efficiently converted into Tregs. To date, this is the most efficient and reliable protocol for generating large numbers of suppressive CD4+ Tregs, which can be used for further biological study and developed for antigen-specific cellular therapy applications

In vivo thrombin activity in the diatom Phaeodactylum tricornutum: Biotechnological insights

Abstract Diatoms are responsible for 20% of global carbon dioxide fixation and have significant potential in various biotechnological and industrial applications. Recently, the pennate diatom Phaeodactylum tricornutum has emerged as a prominent platform organism for metabolic engineering and synthetic biology. The availability of its genome sequence has facilitated the development of new bioengineering tools. In this study, we used in silico analyses to identify sequences potentially encoding thrombin-like proteins, which are involved in recognizing and cleaving the thrombin sequence LVPRGS in P. tricornutum. Protein structure prediction and docking studies indicated a similar active site and ligand positioning compared to characterized human and bovine thrombin. The evidence and efficiency of the cleavage were determined in vivo using two fusion-protein constructs that included YFP to measure expression, protein accumulation, and cleavage. Western blot analysis revealed 50–100% cleavage between YFP and N-terminal fusion proteins. Our findings suggest the existence of a novel thrombin-like protease in P. tricornutum. This study advances the application of diatoms for the synthesis and production of complex proteins and enhances our understanding of the functional role of these putative thrombin sequences in diatom physiology. Key points: • Protein structure predictions reveal thrombin-like active sites in P. tricornutum. • Validated cleavage efficiency of thrombin-like protease on fusion proteins in vivo. • Study advances bioengineering tools for diatom-based biotechnological applications

Biochemical Analyses of Bioactive Extracts from Plants Native to Lampedusa, Sicily Minor Island

Major threats to the human lifespan include cancer, infectious diseases, diabetes, mental degenerative conditions and also reduced agricultural productivity due to climate changes, together with new and more devastating plant diseases. From all of this, the need arises to find new biopesticides and new medicines. Plants and microorganisms are the most important sources for isolating new metabolites. Lampedusa Island host a rich contingent of endemic species and subspecies. Seven plant species spontaneously growing in Lampedusa, i.e., Atriplex halimus L. (Ap), Daucus lopadusanus Tineo (Dl), Echinops spinosus Fiori (Es) Glaucium flavum Crantz (Gf) Hypericum aegypticum L: (Ha), Periploca angustifolia Labill (Pa), and Prasium majus L. (Pm) were collected, assessed for their metabolite content, and evaluated for potential applications in agriculture and medicine. The HPLC-MS analysis of n-hexane (HE) and CH2Cl2 (MC) extracts and the residual aqueous phases (WR) showed the presence of several metabolites in both organic extracts. Crude HE and MC extracts from Dl and He significantly inhibited butyrylcholinesterase, as did WR from the extraction of Dl and Pa. HE and MC extracts showed a significant toxicity towards hepatocarcinoma Huh7, while Dl, Ha and Er HE extracts were the most potently cytotoxic to ileocecal colorectal adenocarcinoma HCT-8 cell lines. Most extracts showed antiviral activity. At the lowest concentration tested (1.56 μg/mL), Dl, Gf and Ap MC extracts inhibited betacoronavirus HCoV-OC43 infection by> 2 fold, while the n-hexane extract of Pm was the most potent. In addition, at 1.56 μg/mL, potent inhibition (>10 fold) of dengue virus was detected for Dl, Er, and Pm HE extracts, while Pa and Ap MC extracts dampened infections to undetectable levels. Regarding to phytotoxicity, MC extracts from Er, Ap and Pm were more effective in inhibiting tomato rootlet elongation; the same first two extracts also inhibited seed cress germination while its radicle elongation, due to high sensitivity, was affected by all the extracts. Es and Gf MC extracts also inhibited seed germination of Phelipanche ramosa. Thus, we have uncovered that many of these Lampedusa plants displayed promising biopesticide, antiviral, and biological properties

Multifactorial interaction and influence of culture conditions on yellow fluorescent protein production in Phaeodactylum tricornutum

Abstract Phaeodactylum tricornutum is a promising host for light-driven synthesis of heterologous proteins. However, the marine cold-water environment and alkaline-acidic pH shifts in the culture, necessitated by the diatom's growth requirements. In this study, we analyzed the influence of growth condition on maturation and dynamics of the yellow fluorescent protein (YFP) in episomal-transformant P. tricornutum. A mathematical model was developed to detect the parameters that affect biomass and YFP production. Optimized conditions increased YFP mean fluorescence intensity (MFI) per cell by 4.2-fold (3.6 ± 0.6 to 15.4 ± 1.1) and total protein levels in the culture by 1.8-fold (123 ± 4 to 219 ± 9 µg L−1), without affecting biomass. YFP stability studies in P. tricornutum showed that the ubiquitin–proteasome system contributes the degradation of the recombinant protein, whereas newly synthesized YFP remains stable for up to 12 h. This optimization provides insights into the fluorescent protein-based heterologous production in diatoms

Navigating Amaryllidaceae alkaloids: Bridging gaps and charting biosynthetic territories

Abstract Amaryllidaceae alkaloid (AA) biosynthesis has garnered significant attention in recent years, particularly with the commercialization of galanthamine as a treatment for the symptoms of Alzheimer’s disease. A significant amount of research work over the last eight decades has focused on the understanding of AA biosynthesis, starting from early radiolabelling studies to recent multi-omics analysis with modern biotechnological advancements. Those studies enabled the identification of hundreds of metabolites, the characterization of biochemical pathways, and an understanding of the environmental stimuli and of the molecular regulation of these pharmaceutically and agriculturally important metabolites. Despite numerous studies, there remain significant gaps in understanding the biosynthesis of AAs in Amaryllidaceae plants. As such, further research is needed to fully elucidate the metabolic pathways and facilitate their production. This review aims to provide a comprehensive summary of the current state of knowledge on AA biosynthesis, from elicitation of expression of transcription factors in the cell nucleus to alkaloid transport in the apoplast, and to highlight the challenges that need to be overcome for further advancement