Antitumoral activity of different Amaryllidaceae alkaloids: In vitro and in silico assays.

ETHNOPHARMACOLOGY RELEVANCE: The plants of Amaryllidaceae family, such as Amaryllis belladonna L., have been used as herbal remedies for thousands of years to address various disorders, including diseases that might today be identified as cancer. AIM OF THE STUDY: The objective of this work was to evaluate the potential of three Amaryllidaceae alkaloids against four cancer cell lines. MATERIAL AND METHODS: The alkaloids lycorine, 1-O-acetylcaranine, and montanine were evaluated in vitro against colon adenocarcinoma cell line (HCT-116) and breast carcinoma cell lines (MCF-7, MDAMB231, and Hs578T). Computational experiments (target prediction and molecular docking) were conducted to gain a deeper comprehension of possible interactions between these alkaloids and potential targets associated with these tumor cells. RESULTS: Montanine presented the best results against HCT-116, MDAMB231, and Hs578T cell lines, while lycorine was the most active against MCF-7. In alignment with the target prediction outcomes and existing literature, four potential targets were chosen for the molecular docking analysis: CDK8, EGFR, ER-alpha, and dCK. The docking scores revealed two potential targets for the alkaloids with scores similar to co-crystallized inhibitors and substrates: CDK8 and dCK. A visual analysis of the optimal docked configurations indicates that the alkaloids may interact with some key residues in contrast to the other docked compounds. This observation implies their potential to bind effectively to both targets. CONCLUSIONS: In vitro and in silico results corroborate with data literature suggesting the Amaryllidaceae alkaloids as interesting molecules with antitumoral properties, especially montanine, which showed the best in vitro results against colorectal and breast carcinoma. More studies are necessary to confirm the targets and pharmaceutical potential of montanine against these cancer cell lines.

A mini-review of the anti-SARS-CoV-2 potency of Amaryllidaceae alkaloids.

BACKGROUND: Nature has perennially served as an infinite reservoir of diverse chemicals with numerous applications benefiting humankind. In recent years, due to the emerging COVID-19 pandemic, there has been a surge in studies on repurposing natural products as anti-SARS-CoV-2 agents, including plant-derived substances. Among all types of natural products, alkaloids remain one of the most important groups with various known medicinal values. The current investigation focuses on Amaryllidaceae alkaloids (AAs) since AAs have drawn significant scientific attention as anti-SARS-CoV-2 agents over the past few years. PURPOSE AND STUDY DESIGN: This study serves as a mini-review, summarizing recent advances in studying the anti-SARS-CoV-2 potency of AAs, covering two aspects: structure-activity relationship and mechanism of action (MOA). METHODS: The study covers the period from 2019 to 2023. The information in this review were retrieved from common databases including Web of Science, ScienceDirect, PubMed and Google scholar. Reported anti-SARS-CoV-2 potency, cytotoxicity and possible biological targets of AAs were summarized and classified into different skeletal subclasses. Then, the structure-activity relationship (SAR) was explored, pinpointing the key pharmacophore-related structural moieties. To study the mechanism of action of anti-SARS-CoV-2 AAs, possible biological targets were discussed. RESULTS: In total, fourteen research articles about anti-SARS-CoV-2 was selected. From the SAR point of view, four skeletal subclasses of AAs (lycorine-, galanthamine-, crinine- and homolycorine-types) appear to be promising for further investigation as anti-SARS-CoV-2 agents despite experimental inconsistencies in determining in vitro half maximal inhibitory effective concentration (EC50). Narciclasine, haemanthamine- and montanine-type skeletons were cytotoxic and devoid of anti-SARS-CoV-2 activity. The lycorine-type scaffold was the most structurally diverse in this study and preliminary structure-activity relationships revealed the crucial role of ring C and substituents on rings A, C and D in its anti-SARS-CoV-2 activity. It also appears that two enantiomeric skeletons (haemanthamine- and crinine-types) displayed opposite activity/toxicity profiles regarding anti-SARS-CoV-2 activity. Pharmacophore-related moieties of the haemanthamine/crinine-type skeletons were the substituents on rings B, C and the dioxymethylene moiety. All galanthamine-type alkaloids in this study were devoid of cytotoxicity and it appears that varying substituents on rings C and D could enhance the anti-SARS-CoV-2 potency. Regarding MOAs, initial experimental results suggested Mpro and RdRp as possible viral targets. Dual functionality between anti-inflammatory activity on host cells and anti-SARS-CoV-2 activity on the SARS-CoV-2 virus of isoquinoline alkaloids, including AAs, were suggested as the possible MOAs to alleviate severe complications in COVID-19 patients. This dual functionality was proposed to be related to the p38 MAPK signaling pathway. CONCLUSION: Overall, Amaryllidaceae alkaloids appear to be promising for further investigation as anti-SARS-CoV-2 agents. The skeletal subclasses holding the premise for further investigation are lycorine-, crinine-, galanthamine- and homolycorine-types.

Identifying and validating potential therapeutic targets for septic heart failure and the cardioprotective effects of lycorine.

BACKGROUND: Septic heart failure has been recognized as a puzzle since antiquity and poses a major challenge to modern medicine. Our previous work has demonstrated the potential effects of lycorine (LYC) on sepsis and septic myocardial injury. Nonetheless, further exploration is needed to elucidate the underlying cellular and molecular mechanisms. METHODS: In this study, we conducted transcriptome analysis and weighted gene co-expression network analysis (WGCNA) to identify the key genes and reveal the mechanism of LYC against septic heart failure. PURPOSE: This study aims to apply bioinformatic analysis and experimental validations to explore the protective effects and underlying mechanism of LYC on the cecal ligation and puncture (CLP)-induced sepsis model mice. RESULTS: Transcriptome analysis revealed the differentially expressed genes (DEGs) following LYC treatment. WGCNA analysis identified gene modules associated with LYC-mediated protection, with BCL3 emerging as a core gene within these modules. Notably, BCL3 was an overlapping gene of DEGs and WGCNA core genes induced by LYC treatment, and is highly negatively correlated with cardiac function indicator. In vivo and in vitro study further prove that LYC exerted a protective effect against septic myocardial injury through inhibiting BCL3. BCL3 siRNA ameliorated LPS-induced cardiac injury and inflammation, while BCL3 overexpression reversed the protective effect of LYC against LPS injury. CONCLUSION: In summary, our findings demonstrate the significant attenuation of septic myocardial disorder by LYC, with the identification of BCL3 as a pivotal target gene. This study is the first to report the role of BCL3 in sepsis and septic myocardial injury. Furthermore, the strategy for hub genes screening used in our study facilitates a comprehensive exploration of septic targets and reveals the potential targets for LYC effect. These findings may offer a new therapeutic strategy for the management of septic heart failure, highlighting the cardioprotective effect of LYC as adjunctive therapy for sepsis management.

Characterization of cinnamate 4-hydroxylase (CYP73A) and p-coumaroyl 3'-hydroxylase (CYP98A) from Leucojum aestivum, a source of Amaryllidaceae alkaloids.

Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans-cinnamic acid, p-coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans-cinnamate 4-hydroxylase (LaeC4H) and p-coumaroyl shikimate/quinate 3'-hydroxylase (LaeC3'H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase (LaeAPX/C3H). Although LaeAPX/C3H consumed p-coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing LaeC4H converted trans-cinnamate to p-coumaric acid, whereas LaeC3'H catalyzed specifically the 3-hydroxylation of p-coumaroyl shikimate, rather than of free p-coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications.

Identification of transcription factor genes responsive to MeJA and characterization of a LaMYC2 transcription factor positively regulates lycorine biosynthesis in Lycoris aurea.

Jasmonates (JAs) are among the main phytohormones, regulating plant growth and development, stress responses, and secondary metabolism. As the major regulator of the JA signaling pathway, MYC2 also plays an important role in plant secondary metabolite synthesis and accumulation. In this study, we performed a comparative transcriptome analysis of Lycoris aurea seedlings subjected to methyl jasmonate (MeJA) at different treatment times. A total of 31,193 differentially expressed genes (DEGs) were identified by RNA sequencing. Among them, 732 differentially expressed transcription factors (TFs) comprising 51 TF families were characterized. The most abundant TF family was WRKY proteins (80), followed by AP2/ERF-EFR (67), MYB (59), bHLH (52), and NAC protein (49) families. Subsequently, by calculating the Pearson's correlation coefficient (PCC) between the expression level of TF DEGs and the lycorine contents, 41 potential TF genes (|PCC| >0.8) involved in lycorine accumulation were identified, including 36 positive regulators and 5 negative regulators. Moreover, a MeJA-inducible MYC2 gene (namely LaMYC2) was cloned on the basis of transcriptome sequencing. Bioinformatic analyses revealed that LaMYC2 proteins contain the bHLH-MYC_N domain and bHLH-AtAIB_like motif. LaMYC2 protein is localized in the cell nucleus, and can partly rescue the MYC2 mutant in Arabidopsis thaliana. LaMYC2 protein could interact with most LaJAZs (especially LaJAZ3 and LaJAZ4) identified previously. Transient overexpression of LaMYC2 increased lycorine contents in L. aurea petals, which might be associated with the activation of the transcript levels of tyrosine decarboxylase (TYDC) and phenylalanine ammonia lyase (PAL) genes. By isolating the 887-bp-length promoter fragment upstream of the start codon (ATG) of LaTYDC, we found several different types of E-box motifs (CANNTG) in the promoter of LaTYDC. Further study demonstrated that LaMYC2 was indeed able to bind the E-box (CACATG) present in the LaTYDC promoter, verifying that the pathway genes involved in lycorine biosynthesis could be regulated by LaMYC2, and that LaMYC2 has positive roles in the regulation of lycorine biosynthesis. These findings demonstrate that LaMYC2 is a positive regulator of lycorine biosynthesis and may facilitate further functional research of the LaMYC2 gene, especially its potential regulatory roles in Amaryllidaceae alkaloid accumulation in L. aurea.

Lycorine inhibits Ang II-induced heart remodeling and inflammation by suppressing the PI3K-AKT/NF-κB pathway.

BACKGROUND: Ang II induces hypertensive heart failure (HF) via hemodynamic and non-hemodynamic actions. Lycorine (LYC) is an alkaloid derived from Lycoris bulbs, and it possesses anti-cardiovascular disease-related activities. Herein, we explored the potential LYC-mediated regulation of Ang II-induced HF. METHODS: Over 4 weeks, we established a hypertensive HF mouse model by infusing Ang II into C57BL/6 mice using a micro-osmotic pump. For the final two weeks, mice were administered LYC via intraperitoneal injection. The LYC signaling network was then deduced using RNA sequencing. RESULTS: LYC administration strongly suppressed hypertrophy, myocardial fibrosis, and cardiac inflammation. As a result, it minimized heart dysfunction while causing no changes in blood pressure. The Nuclear Factor kappa B (NF-κB) network/phosphoinositol-3-kinase (PI3K)-protein kinase B (AKT) was found to be a major modulator of LYC-based cardioprotection using RNA sequencing study. We further confirmed that in cultured cardiomyocytes and mouse hearts, LYC reduced the inflammatory response and downregulated the Ang II-induced PI3K-AKT/NF-κB network. Moreover, PI3K-AKT or NF-κB axis depletion in cardiomyocytes completely abrogated the anti-inflammatory activities of LYC. CONCLUSION: Herein, we demonstrated that LYC safeguarded hearts in Ang II -stimulated mice by suppressing the PI3K-AKT/NF-κB-induced inflammatory responses. Given the evidence mentioned above, LYC is a robust therapeutic agent for hypertensive HF.

Dynamics of alkaloid accumulation in Narcissus cv. Hawera: a source of Sceletium-type alkaloids.

The Sceletium-type alkaloids, known for their anxiolytic and antidepressant activities, have been recently found to be biosynthesized in Narcissus cv. Hawera, which is largely used as an ornamental plant. An alkaloid fraction enriched with Sceletium-type alkaloids from the plant has shown promising antidepressant and anxiolytic activities. In the present study, qualitative and quantitative analyses of the alkaloids in the plant organs were performed during one vegetation season by GC-MS. The alkaloid pattern and total alkaloid content was found to depend strongly on the stage of development and plant organ. The alkaloid content of bulbs was found to be highest during the dormancy period and lowest in sprouting bulbs. The leaves showed the highest alkaloid content during the intensive vegetative growth and lowest during flowering. In total, 13 alkaloids were detected in the methanol extracts of Narcissus cv. Hawera, six Sceletium-type and seven typical Amaryllidaceae alkaloids. Major alkaloids in the alkaloid pattern were lycorine, 6-epi-mesembrenol, mesembrenone, sanguinine, and galanthamine. The leaves of flowering plants were found to have the highest amount of 6-epi-mesembrenol. Mesembrenone was found to be dominant alkaloid in the leaves of sprouting bulbs and in the flowers. Considering the biomass of the plant, the dormant bulbs are the best source of alkaloid fractions enriched with 6-epi-mesembrenol. The flowers and the young leaves can be used for preparation of alkaloid fractions enriched with mesembrenone. The results indicates that Narcissus cv. Hawera is an emerging source of valuable bioactive compounds and its utilization can be extended as a medicinal plant.

Preparation and Evaluation of Folate Modified PEG-PLLA Nanoparticles Loaded with Lycorine for Glioma Treatment.

Lycorine is a kind of natural active ingredient with a strong antitumor effect. In this study, folate ligand-conjugated polyethylene glycol-block-poly(l-lactide) (PEG-PLLA) nanoparticles (FA-PEG-PLLA NPs) were designed to deliver lycorine to enhance its anti-glioma activity. The successful preparation of the FA-PEG-PLLA polymer was confirmed by 1H-NMR, FT-IR, XRD, TGA, and DSC. The optimal formulation for LYC@FA-PEG-PLLA NPs was determined by response surface analysis as follows: sodium dodecyl sulfate (SDS) of 1%, carrier material of 0.03 g, organic phase volume of 3 mL, and ultrasonic power of 20%. The LYC@FA-PEG-PLLA NPs exhibited an encapsulation efficiency of 83.58% and a particle size of 49.71 nm, demonstrating good stability. Hemolysis experiments, MTT assays, and cell scratch assays revealed excellent biocompatibility of FA-PEG-PLLA and superior anti-glioma activity of LYC@FA-PEG-PLLA NPs compared to the raw drug. Additionally, cell apoptosis assays, ROS experiments, and western blot analysis demonstrated that LYC@FA-PEG-PLLA NPs contributed to cell apoptosis by inducing ROS generation and increasing the expression of NF-κB inhibitory protein IκBα. These results suggested that LYC@FA-PEG-PLLA NPs hold promise for glioma treatment.

Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme.

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC50 = 20 µM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4'-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.

Molecular Docking and GC/MS-Based Approach for Identification of Anxiolytic Alkaloids from Griffinia (Amaryllidaceae) Species in a Zebrafish Model.

Griffinia gardneriana Ravenna, Griffinia liboniana Morren and Griffinia nocturna Ravenna (Amarillydaceae) are bulbous plants found in tropical regions of Brazil. Our work aimed to determine the alkaloid profiles of Griffinia spp. and evaluate their anxiolytic potential through in vivo and in silico assays. The plants grown in greenhouses were dried and their ground bulbs were subjected to liquid-liquid partitions, resulting in alkaloid fractions that were analyzed by gas chromatography coupled to mass spectrometry (GC-MS). Anxiolytic activity was evaluated in zebrafish (Danio rerio) through intraperitoneal injection at doses of 40, 100 and 200 mg/kg in light-dark box test. GC-MS analyses revealed 23 alkaloids belonging to different skeleton types: lycorine, homolychorine, galanthamine, crinine, haemanthamine, montanine and narcisclasine. The chemical profiles were relatively similar, presenting 8 alkaloids common to the three species. The major component for G. gardneriana and G. liboniana was lycorine, while G. nocturna consisted mainly of anhydrolycorine. All three alkaloid fractions demonstrated anxiolytic effect. Furthermore, pre-treatment with diazepam and pizotifen drugs was able to reverse the anxiolytic action, indicating involving the GABAergic and serotonergic receptors. Molecular docking showed that the compounds vittatine, lycorine and 11,12-dehydro-2-methoxyassoanine had high affinity with both receptors, suggesting them to be responsible for the anxiolytic effect.

Lycorine and homolycorine derivatives for chemo-sensitizing resistant human ovarian adenocarcinoma cells.

BACKGROUND: Multidrug resistance is the major obstacle to cancer chemotherapy. Modulation of P-glycoprotein and drug combination approaches have been considered important strategies to overcome drug resistance. PURPOSE: Aiming at generating a small library of Amaryllidaceae-type alkaloids to overcome drug resistance, two major alkaloids, isolated from Pancratium maritimum, lycorine (1), and 2α-10bα-dihydroxy-9-O-demethylhomolycorine (2), were derivatized, giving rise to nineteen derivatives (3 - 21). METHODS: The main chemical transformation of lycorine resulted from the cleavage of ring E of the diacetylated lycorine derivative (3) to obtain compounds that have carbamate and amine functions (5 - 16), while acylation of compound 2 provided derivatives 17 - 21. Compounds 1 - 21 were evaluated for their effects on cytotoxicity, and drug resistance reversal, using resistant human ovarian carcinoma cells (HOC/ADR), overexpressing P-glycoprotein (P-gp/ABCB1), as model. RESULTS: Excluding lycorine (1) (IC50 values of 1.2- 2.5 µM), the compounds were not cytotoxic or showed moderate/weak cytotoxicity. Chemo-sensitization assays were performed by studying the in vitro interaction between the compounds and the anticancer drug doxorubicin. Most of the compounds have shown synergistic interactions with doxorubicin. Compounds 5, 6, 9 - 14, bearing both carbamate and aromatic amine moieties, were found to have the highest sensitization rate, reducing the dose of doxorubicin 5-35 times, highlighting their potential to reverse drug resistance in combination chemotherapy. Selected compounds (4 - 6, 9 - 14, and 21), able of re-sensitizing resistant cancer cells, were further evaluated as P-gp inhibitors. Compound 11, which has a para­methoxy-N-methylbenzylamine moiety, was the strongest inhibitor. In the ATPase assay, compounds 9-11 and 13 behaved as verapamil, suggesting competitive inhibition of P-gp. At the same time, none of these compounds affected P-gp expression at the mRNA or protein level. CONCLUSIONS: This study provided evidence of the potential of Amaryllidaceae alkaloids as lead candidates for the development of MDR reversal agents.

Ligand fishing approach to explore Amaryllidaceae alkaloids as potential antiviral candidates targeting SARS-CoV-2 Nsp4.

Ligand fishing, also described as affinity-based assay, represents a convenient and efficient approach to separate potential ligands from complex matrixes or chemical libraries. This approach contributes to the identification of lead compounds that can bind to a specific target. In the context of COVID-19, the search for novel therapeutic agents is crucial. Small molecule-based antiviral drugs, such as Amaryllidaceae alkaloids, have been described as potential candidates because they can inhibit RNA viruses. Among various SARS-CoV-2 proteins, Nsp3, Nsp4, and Nsp6 play a crucial role in the pathogenicity of the virus and are attractive targets for developing COVID-19 treatments. These proteins are responsible for the replication/transcription complex (RTC) within double-membrane vesicles (DMVs), and their inhibition disrupts the virus's infectious cycle. Herein, we have successfully expressed and immobilized the SARS-CoV-2 Nsp4 protein on magnetic beads (Nsp4-MBs) and employed a ligand fishing assay to screen a collection of ten Amaryllidaceae-based alkaloids and applied to Hippeastrum aulicum extract. Remarkably, four out of ten alkaloids, namely 2-α-7-dimethoxyhomolycorine (6), haemanthamine (5), albomaculine (8), and tazettine (9), exhibited selective affinities for Nsp4. Albomaculine (8) and haemanthamine (5) were also identified from extract by the affinity assay. These findings highlight the potential of these alkaloids as model compounds for future drug discovery studies aimed at developing therapeutic interventions against SARS-CoV-2 infections.

Bruceine D and Narclasine inhibit the proliferation of breast cancer cells and the prediction of potential drug targets.

BACKGROUND: Breast cancer is one of the most common female malignancies. This study explored the underlying mechanism through which the two plant compounds (Brucaine D and Narclasine) inhibited the proliferation of breast cancer cells. OBJECTIVE: The purpose of this study was to explore the effect of Brucaine D and Narclasine on breast cancer development and their potential drug targets. METHODS: GSE85871 dataset containing 212 samples and the hallmark gene set "h.all.v2023.1.Hs.symbols.gmt" were downloaded from the Gene Expression Omnibus (GEO) database and the Molecular Signatures Database (MSigDB) database, respectively. Principal component analysis (PCA) was applied to classify clusters showing similar gene expression pattern. Single sample gene set enrichment analysis (ssGSEA) was used to calculate the hallmark score for different drug treatment groups. The expressions of genes related to angiogenesis, glycolysis and cell cycle were detected. Protein-protein interaction (PPI) network analysis was performed to study the interaction of the hub genes. Then, HERB database was employed to identify potential target genes for Narclasine and Bruceine D. Finally, in vitro experiments were conducted to validate partial drug-target pair. RESULTS: PCA analysis showed that the significant changes in gene expression patterns took place in 6 drugs treatment groups (Narciclasine, Bruceine D, Japonicone A, 1beta-hydroxyalatolactone, Britanin, and four mixture drugs) in comparison to the remaining drug treatment groups. The ssGSEA pathway enrichment analysis demonstrated that Narciclasine and Bruceine treatments had similar enriched pathways, for instance, suppressed pathways related to angiogenesis, Glycolysis, and cell cycle, etc.. Further gene expression analysis confirmed that Narciclasine and Bruceine had a strong ability to inhibit these cell cycle genes, and that MYC, CHEK2, MELK, CDK4 and EZH2 were closely interacted with each other in the PPI analysis. Drug target prediction revealed that Androgen Receptor (AR) and Estrogen Receptor 1 (ESR1) were the targets for Bruceine D, and Cytochrome P450 3A4 enzyme (CYP3A4) was the target for Narciclasine. Cell experiments also confirmed the connections between Narciclasine and CYP3A4. CONCLUSION: The present study uncovered that Narciclasine and Bruceine D could inhibit the growth of breast cancer and also predicted the potential targets for these two drugs, providing a new therapeutic direction for breast cancer patients.

Molecular mechanism of lycorine in the treatment of glioblastoma based on network pharmacology and molecular docking.

Lycorine is a naturally active alkaloid that has been shown to have inhibitory effects on a variety of cancers. However, the underlying mechanism of lycorine in the treatment of glioblastoma (GBM) is unclear. In this study, we investigated the mechanism of lycorine in the treatment of GBM based on network pharmacology and molecular docking. Lycorine-related targets overlapped with GBM-related targets to obtain intersections that represent potential anti-GBM targets for lycorine. The protein-protein interaction (PPI) network was constructed using the STRING online database and analyzed by Cytoscape software, and 10 key target genes (AKT1, SRC, HSP90AA1, HRAS, MMP9, BCL2L1, IGF1, MAPK14, STAT1, and KDR) were obtained, which played an important role in the therapeutic effect of lycorine on GBM. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that lycorine acts on GBM by multiple pathways, including inducing apoptosis and reactive oxygen species production. The molecular docking results showed that lycorine had strong binding efficiency with the 10 key genes. In addition, we found that the use of lycorine-induced apoptosis in U-87 MG glioblastoma cells. Here, the mechanism of action of lycorine against GBM was elucidated and verified by experiments, which provided evidence support for its clinical application.

Unveiling Amaryllidaceae alkaloids: from biosynthesis to antiviral potential - a review.

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.

Amaryllidaceae alkaloids from the bulbs of Crinum latifolium L. and their cholinesterase inhibitory activities.

Eleven previously undescribed Amaryllidaceae alkaloids, crinalatifolines A-K (1-11), and two first naturally occurring alkaloids, dihydroambelline (12) and N-demethyldihydrogalanthamine (13), were isolated from the bulbs of Crinum latifolium L. Additionally, thirty-seven known alkaloids and one alkaloid artifact were also isolated from this plant species. Their structures and absolute configurations were elucidated using extensive spectroscopic techniques, including IR, NMR, MS, and ECD. Evaluations of the cholinesterase inhibitory activities of most of these compounds were conducted. Among the tested compounds, ungeremine exhibited the highest potency against acetylcholinesterase and butyrylcholinesterase, with the IC50 values of 0.10 and 1.21 µM, respectively. These values were 9.4- and 2.4-fold more potent than the reference drug galanthamine.

Interplay between Amaryllidaceae alkaloids, the bacteriome and phytopathogens in Lycoris radiata.

Alkaloids are a large group of plant secondary metabolites with various structures and activities. It is important to understand their functions in the interplay between plants and the beneficial and pathogenic microbiota. Amaryllidaceae alkaloids (AAs) are unique secondary metabolites in Amaryllidaceae plants. Here, we studied the interplay between AAs and the bacteriome in Lycoris radiata, a traditional Chinese medicinal plant containing high amounts of AAs. The relationship between AAs and bacterial composition in different tissues of L. radiata was studied. In vitro experiments revealed that AAs have varying levels of antimicrobial activity against endophytic bacteria and pathogenic fungi, indicating the importance of AA synthesis in maintaining a balance between plants and beneficial/pathogenic microbiota. Using bacterial synthetic communities with different compositions, we observed a positive feedback loop between bacteria insensitive to AAs and their ability to increase accumulation of AAs in L. radiata, especially in leaves. This may allow insensitive bacteria to outcompete sensitive ones for plant resources. Moreover, the accumulation of AAs enhanced by insensitive bacteria could benefit plants when challenged with fungal pathogens. This study highlights the functions of alkaloids in plant-microbe interactions, opening new avenues for designing plant microbiomes that could contribute to sustainable agriculture.

Auxin and light-mediated regulation of growth, morphogenesis, and alkaloid biosynthesis in Crinum x powellii 'Album' callus.

Crinum x powellii 'Album' belongs to the Amaryllidaceae medicinal plant family that produces a range of structurally diverse alkaloids with potential therapeutic properties. The optimal conditions for in vitro tissue growth, morphogenesis, and alkaloid biosynthesis remain unclear. Auxin and light play critical roles in regulating plant growth, development, and alkaloid biosynthesis in several Amaryllidaceae plants. Here, we have succeeded in showing, for the first time, that the combination of auxin and light significantly influence C. x powellii "Album" in vitro tissue growth, survival, and morphogenesis compared to individual treatments. Furthermore, this combination also upregulates the expression of alkaloid biosynthetic genes and led to an increase in the content of certain alkaloids, suggesting a positive impact on the defense and therapeutic potential of the calli. Our findings provide insights into the regulation of genes involved in alkaloid biosynthesis in C. x powellii "Album" callus and underline the potential of auxin and light as tools for enhancing their production in plants. This study provides a foundation for further exploration of C. x powellii "Album" calli as a sustainable source of bioactive alkaloids for pharmaceutical and agricultural applications. Furthermore, this study paves the way to the discovery of the biosynthetic pathway of specialized metabolites from C. x powellii "Album", such as cherylline and lycorine.

Lycorine inhibits pancreatic cancer cell growth and neovascularization by inducing Notch1 degradation and downregulating key vasculogenic genes.

Pancreatic cancer is highly metastatic and lethal with an increasing incidence globally and a 5-year survival rate of only 8%. One of the factors contributing to the high mortality is the lack of effective drugs in the clinical setting. We speculated that effective compounds against pancreatic cancer exist in natural herbs and explored active small molecules among traditional Chinese medicinal herbs. The small molecule lycorine (MW: 323.77) derived from the herb Lycoris radiata inhibited pancreatic cancer cell growth with an IC50 value of 1 µM in a concentration-dependent manner. Lycorine markedly reduced pancreatic cancer cell viability, migration, invasion, neovascularization, and gemcitabine resistance. Additionally, lycorine effectively suppressed tumor growth in mouse xenograft models without obvious toxicity. Pharmacological studies revealed that the levels and half-life of Notch1 oncoprotein in the pancreatic cancer cells Panc-1 and Patu8988 were notably reduced. Moreover, the expression of the key vasculogenic genes Semaphorin 4D (Sema4D) and angiopoietin-2 (Ang-2) were also significantly inhibited by lycorine. Mechanistically, lycorine strongly triggered the degradation of Notch1 oncoprotein through the ubiquitin-proteasome system. In conclusion, lycorine effectively inhibits pancreatic cancer cell growth, migration, invasion, neovascularization, and gemcitabine resistance by inducing degradation of Notch1 oncoprotein and downregulating the key vasculogenic genes Sema4D and Ang-2. Our findings provide a new therapeutic candidate and treatment strategy against pancreatic cancer.

Antiproliferative activity and apoptosis-inducing mechanism of Amaryllidaceae alkaloid montanine on A549 and MOLT-4 human cancer cells.

The isoquinoline alkaloids found in Amaryllidaceae are attracting attention due to attributes that can be harnessed for the development of new drugs. The possible molecular mechanisms by which montanine exerts its inhibitory effects against cancer cells have not been documented. In the present study, montanine, manthine and a series of 15 semisynthetic montanine analogues originating from the parent alkaloid montanine were screened at a single test dose of 10 µM to explore their cytotoxic activities against a panel of eight cancer cell lines and one non-cancer cell line. Among montanine and its analogues, montanine and its derivatives 12 and 14 showed the highest cytostatic activity in the initial single-dose screening. However, the native montanine exhibited the greatest antiproliferative activity against cancer cells, with a lower mean IC50 value of 1.39 µM, compared to the displayed mean IC50 values of 2.08 µM for 12 and 3.57 µM for 14. Montanine exhibited the most potent antiproliferative activity with IC50 values of 1.04 µM and 1.09 µM against Jurkat and A549 cell lines, respectively. We also evaluated montanine's cytotoxicity and cell death mechanisms. Our results revealed that montanine triggered apoptosis of MOLT-4 cells via caspase activation, mitochondrial depolarisation and Annexin V/PI double staining. The Western blot results of MOLT-4 cells showed that the protein levels of phosphorylated Chk1 Ser345 were upregulated with increased montanine concentrations. Our findings provide new insights into the mechanisms underlying the cytostatic, cytotoxic and pro-apoptotic activities of montanine alkaloids in lung adenocarcinoma A549 and leukemic MOLT-4 cancer cell types.