Tamarix aphylla derived metabolites ameliorate indomethacin-induced gastric ulcers in rats by modulating the MAPK signaling pathway, alleviating oxidative stress and inflammation: In vivo study supported by pharmacological network analysis.

Nature has proven to be a treasure resource of bioactive metabolites. In this regard, Tamarix aphylla (F. Tamaricaceae) leaves crude extract was investigated for its gastroprotective effect against indomethacin-induced damage to the gastric mucosa. Additionally, phytochemical investigation of the methanolic extract afforded eight flavonoids' derivatives (1-8). On pharmacology networking study, the isolated compounds identified 123 unique targets where only 45 targets were related to peptic ulcer conditions, these 45 targets include 11 targets specifically correlate to gastric ulcer. The protein-protein interaction defined the PTGS2 gene as one of the highly interacted genes and the complete pharmacology network defined the PTGS2 gene as the most represented gene. The top KEGG signaling pathways according to fold enrichment analysis was the EGFR tyrosine kinase inhibitor resistance pathway. As a result, these findings highlighted the significance of using T. aphylla leaves crude extract as an anti-gastric ulcer candidate, which provides a safer option to chemical antisecretory medicines, which are infamous for their negative side effects. Our findings have illuminated the potent anti-inflammatory and antioxidant effects of T. aphylla, which are likely mediated by suppressing IL-1ß, IL-6, TNF-α, and MAPK signaling pathways, without compromising gastric acidity.

Naphthylisoquinoline alkaloids: novel agents against the causative pathogens of eumycetoma and actinomycetoma-en route to broad-spectrum antimycetomal drugs.

Mycetoma is a devastating neglected tropical infection of the subcutaneous tissues. It is caused by fungal and bacterial pathogens recognized as eumycetoma and actinomycetoma, respectively. Mycetoma treatment involves diagnosing the causative microorganism as a prerequisite to prescribing a proper medication. Current therapy of fungal eumycetoma causative agents, such as Madurella mycetomatis, consists of long-term antifungal medication with itraconazole followed by surgery, yet with usually unsatisfactory clinical outcomes. Actinomycetoma, on the contrary, usually responds to treatment with co-trimoxazole and amikacin. Therefore, there is a pressing need to discover novel broad-spectrum antimicrobial agents to circumvent the time-consuming and costly diagnosis. Using the resazurin assay, a series of 23 naphthylisoquinoline (NIQ) alkaloids and related naphthoquinones were subjected to in vitro screening against two fungal strains of M. mycetomatis and three bacterial strains of Actinomadura madurae and A. syzygii. Seven NIQs, mostly dimers, showed promising in vitro activities against at least one strain of the mycetoma-causative pathogens, while the naphthoquinones did not show any activity. A synthetic NIQ dimer, 8,8'''-O,O-dimethylmichellamine A (18), inhibited all tested fungal and bacterial strains (IC50 = 2.81-12.07 µg/mL). One of the dimeric NIQs, michellamine B (14), inhibited a strain of M. mycetomatis and significantly enhanced the survival rate of Galleria mellonella larvae infected with M. mycetomatis at concentrations of 1 and 4 µg/mL, without being toxic to the uninfected larvae. As a result, broad-spectrum dimeric NIQs like 14 and 18 with antimicrobial activity are considered hit compounds that could be worth further optimization to develop novel lead antimycetomal agents.

Investigation of bioactive components responsible for the antibacterial and anti-biofilm activities of Caroxylon volkensii by LC-QTOF-MS/MS analysis and molecular docking.

Caroxylon volkensii is a wild desert plant of the family Amaranthaceae. This study represents the first report of the metabolomic profiling of C. volkensii by liquid chromatography quadrupole-time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS). The dereplication study of its secondary metabolites led to the characterization of 66 known compounds. These compounds include catecholamines, tyramine derivatives, phenolic acids, triterpenoids, flavonoids, and others. A new tyramine derivative, alongside other known compounds, was reported for the first time in the Amaranthaceae family. The new derivative and the first-reported compounds were putatively identified through MS/MS fragmentation data. Given the notorious taxonomical challenges within the genus Salsola, to which C. volkensii previously belonged, our study could offer a valuable insight into its chemical fingerprint and phylogenetic relationship to different Salsola species. The antibacterial potential of C. volkensii methanolic extract (CVM) against Pseudomonas aeruginosa was screened. The minimum inhibitory concentration (MIC) of CVM ranged from 32 to 256 µg mL-1. The anti-quorum sensing potential of CVM resulted in a decrease in the percentage of strong and moderate biofilm-forming isolates from 47.83% to 17.39%. It revealed a concentration-dependent inhibitory activity on violacein formation by Chromobacterium violaceum. Moreover, CVM exhibited an in vivo protective potential against the killing capacity of P. aeruginosa isolates. A molecular docking study revealed that the quorum-sensing inhibitory effect of CVM can be attributed to the binding of tyramine conjugates, ethyl-p-digallate, and isorhamnetin to the transcriptional global activator LasR.

Wound healing potential of Cystoseira/mesenchymal stem cells in immunosuppressed rats supported by overwhelming immuno-inflammatory crosstalk.

Wound healing, one of the most intricate and dynamic processes of the body, maintains skin integrity following trauma. One of the main issues that still exists is impaired wound healing, particularly for immunosuppressed patients. Recently, natural products from marine environments have been employed in wound-repairing activities. This work investigates the mesenchymal stem cells in the combined capacity of the bone marrow (BMMSC) for wound healing and Cystoseira sp. Algae extract in immunosuppressed rats. High-resolution liquid chromatography / MS investigation of Cystoseira extract revealed the prevalence of fatty acids that have wound-soothing potential. From constructed PPI network for wound healing and further analysis through molecular docking and molecular dynamics (MD) simulation experiments suggested that cystalgerone metabolite may be responsible for the wound healing-promoting effect of Cystoseira extract. According to the CD marker characterization of the BMMSC, 98.21% of them expressed CD90, and 97.1% expressed CD105. Sixteen d after immunity suppression (by 40 mg/kg hydrocortisone daily), an incision was made in the dorsal skin of the rat. The treatments were applied for 16 d and samples were taken from the tested groups on the 8th, 14th, and 16th days. The BMMSCs / Cystoseira group showed significantly improved wound closure, thickness, density of new layers, and skin elasticity than the control group (p < 0.001). The BMMSCs / Cystoseira combination significantly reduced the oxidative indicators, pro-inflammatory cytokines, and immune markers, according to the RT-PCR gene expression study. In order to delve deeper into the complex interconnections among wound healing-related biological targets and pinpoint key factors in this complex process, we engaged in network pharmacology and computational research. Subsequently, we conducted a comprehensive computational analysis, including reverse docking, free energy (ΔG) computation, and molecular dynamics simulations, on the molecular structures of the annotated compounds. The purpose of this investigation was to identify potential new targets for these chemicals as well as any potential interactions they may have with different signaling pathways related to the wound healing process. Our research indicates that the primary compounds of Cystoseira holds potential wound healing therapeutic activity. Although more safety testing and clinical studies are required, the combination has great potential for regenerative medicine and could be a revolutionary advance in the healing of the wounds of immunosuppressed patients.

Jozimine A2, a Dimeric Naphthylisoquinoline (NIQ) Alkaloid, Shows In Vitro Cytotoxic Effects against Leukemia Cells through NF-κB Inhibition.

Naphthylisoquinoline (NIQ) alkaloids are rising as a promising class of secondary metabolites with pharmaceutical potential. NF-κB has already been recognized as a significant modulator of cancer proliferation and drug resistance. We have previously reported the mechanisms behind the cytotoxic effect of dioncophylline A, an NIQ monomer, in leukemia cells. In the current study, we have investigated the cytotoxic effect of jozimine A2, an NIQ dimer, on leukemia cells in comparison to a second, structurally unsymmetric dimer, michellamine B. To this end, molecular docking was applied to predict the binding affinity of the dimers towards NF-κB, which was then validated through microscale thermophoresis. Next, cytotoxicity assays were performed on CCRF-CEM cells and multidrug-resistant CEM/ADR5000 cells following treatment. Transcriptome analysis uncovered the molecular networks affected by jozimine A2 and identified the cell cycle as one of the major affected processes. Cell death modes were evaluated through flow cytometry, while angiogenesis was measured with the endothelial cell tube formation assay on human umbilical vein endothelial cells (HUVECs). The results indicated that jozimine A2 bound to NF-κB, inhibited its activity and prevented its translocation to the nucleus. In addition, jozimine A2 induced cell death through apoptosis and prevented angiogenesis. Our study describes the cytotoxic effect of jozimine A2 on leukemia cells and explains the interactions with the NF-κB signaling pathway and the anticancer activity.

Antiplasmodial potential of phytochemicals from Citrus aurantifolia peels: a comprehensive in vitro and in silico study.

Phytochemical investigation of Key lime (Citrus aurantifolia L., F. Rutaceae) peels afforded six metabolites, known as methyl isolimonate acetate (1), limonin (2), luteolin (3), 3`-hydroxygenkwanin (4), myricetin (5), and europetin (6). The structures of the isolated compounds were assigned by 1D NMR. In the case of limonin (2), further 1- and 2D NMR experiments were done to further confirm the structure of this most active metabolite. The antiplasmodial properties of the obtained compounds against the pathogenic NF54 strain of Plasmodium falciparum were assessed in vitro. According to antiplasmodial screening, only limonin (2), luteolin (3), and myricetin (5) were effective (IC50 values of 0.2, 3.4, and 5.9 µM, respectively). We explored the antiplasmodial potential of phytochemicals from C. aurantifolia peels using a stepwise in silico-based analysis. We first identified the unique proteins of P. falciparum that have no homolog in the human proteome, and then performed inverse docking, ΔGBinding calculation, and molecular dynamics simulation to predict the binding affinity and stability of the isolated compounds with these proteins. We found that limonin (2), luteolin (3), and myricetin (5) could interact with 20S a proteasome, choline kinase, and phosphocholine cytidylyltransferase, respectively, which are important enzymes for the survival and growth of the parasite. According to our findings, phytochemicals from C. aurantifolia peels can be considered as potential leads for the development of new safe and effective antiplasmodial agents.

Apium extract alleviates indomethacin-induced gastric ulcers in rats via modulating the VEGF and IK-κB/NF-κB p65 signaling pathway: insights from in silico and in vivo investigations.

BACKGROUND: Gastric ulcers represent a worldwide health problem, characterized by erosions that affect the mucous membrane of the stomach and may even reach the muscular layer, leading to serious complications. Numerous natural products have been assessed as anti-ulcerogenic agents, and have been considered as new approaches for treatment or prevention of gastric ulcers. The present research investigated the preventive benefits of Apium graveolens L. (Apiaceae), known as celery, seed extract towards indomethacin-induced ulceration of the stomach in rats. METHODS: Metabolomic profiling, employing liquid chromatography coupled to high-resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS), was implemented with the aim of investigating the chemical profile of the seeds. Histopathological analysis of gastric tissues, as well as assessment of numerous inflammatory cytokines and oxidative stress indicators, confirmed the in vivo evaluation. RESULTS: The prior treatment with A. graveolens seed extract resulted in a substantial reduction in the ulcer index when compared to the indomethacin group, indicating an improvement in stomach mucosal injury. Moreover, the gastroprotective effect was demonstrated through examination of the oxidative stress biomarkers which was significantly attenuated upon pre-treatment with A. graveolens seed extract. Vascular endothelial growth factor (VEGF), a fundamental angiogenic factor that stimulates angiogenesis, was markedly inhibited by indomethacin. A. graveolens seed extract restored this diminished level of VEGF. The dramatic reductions in NF-κB protein levels indicate a considerable attenuation of the indomethacin-induced IKκB/NF-κB p65 signaling cascade. These activities were also correlated to the tentatively featured secondary metabolites including, phenolic acids, coumarins and flavonoids, previously evidenced to exert potent anti-inflammatory and antioxidant activities. According to our network pharmacology study, the identified metabolites annotated 379 unique genes, among which only 17 genes were related to gastric ulcer. The PTGS2, MMP2 and PTGS1 were the top annotated genes related to gastric ulcer. The top biological pathway was the VEGF signaling pathway. CONCLUSION: A. graveolens seed extract possesses significant anti-ulcer activity, similar to famotidine, against gastric lesions induced by indomethacin in rats. It is worth highlighting that the extract overcomes the negative effects of conventional chemical anti-secretory drugs because it does not lower stomach acidity.

Cytotoxicity of dioncophylline A and related naphthylisoquinolines in leukemia cells, mediated by NF-κB inhibition, angiogenesis suppression, G2/M cell cycle arrest, and autophagy induction.

BACKGROUND: Inhibition of NF-κB activity represents a strategy to treat acute myeloid leukemia, one of the most lethal leukemia types. Naphthylisoquinolines (NIQs) are cytotoxic alkaloids from lianas of the families Ancistrocladaceae and Dioncophyllaceae, which are indigenous to tropical rainforests. PURPOSE: Uncovering therapeutic possibilities and underlying molecular mechanisms of dioncophylline A and its derivatives towards NF-κB related cellular processes. METHODS: Resazurin-based cell viability assay was performed for dioncophylline A and three derivatives on wild-type CCRF-CEM and multidrug-resistant CEM/ADR5000 cells. Transcriptome analysis was executed to discover cellular functions and molecular networks associated with dioncophylline A treatment. Expression changes obtained by mRNA microarray hybridization were confirmed using qRT-PCR. Molecular docking was applied to predict the affinity of the NIQs with NF-κB. To validate the in silico approach, NF-κB reporter assays were conducted on HEK-Blue™ Null1 cells. Cell death mechanisms and cell cycle arrest were studied using flow cytometry. The potential activity on angiogenesis was evaluated with the endothelial cell tube formation assay on HUVECs using fluorescence microscopy. Intracellular NF-κB location in HEK-Blue™ Null1 cells was visualized with immunofluorescence. Finally, the anti-tumor activity of dioncophylline A was studied by a xenograft zebrafish model in vivo. RESULTS: Our study demonstrated that dioncophylline A and its derivatives exerted potent cytotoxicity on leukemia cells. Using Ingenuity Pathway Analysis, we identified the NF-κB network as the top network, and docking experiments predicted dioncophylline A and two of its derivatives sharing the same binding pocket with the positive control compound, triptolide. Dioncophylline A showed the best inhibitory activity in NF-κB reporter assays compared to its derivatives, caused autophagy rather than apoptosis, and induced G2/M arrest. It also prevented NF-κB translocation from the cytoplasm to the nucleus. Tube formation as an angiogenesis marker was significantly suppressed by dioncophylline A treatment. Finally, the remarkable anti-tumor activity of dioncophylline A was proven in zebrafish in vivo. CONCLUSION: Taken together, we report for the first time the molecular mechanism behind the cytotoxic effect of dioncophylline A on leukemia cells. Dioncophylline A showed strong cytotoxic activity, inhibited NF-κB translocation, significantly affected the NF-κB in silico and in vitro, subdued tube formation, induced autophagy, and exerted antitumor activity in vivo. Our findings enlighten both the cellular functions including the NF-κB signaling pathway and the cytotoxic mechanism affected by dioncophylline A.

Subgenome dominance shapes novel gene evolution in the decaploid pitcher plant Nepenthes gracilis.

Subgenome dominance after whole-genome duplication generates distinction in gene number and expression at the level of chromosome sets, but it remains unclear how this process may be involved in evolutionary novelty. Here we generated a chromosome-scale genome assembly of the Asian pitcher plant Nepenthes gracilis to analyse how its novel traits (dioecy and carnivorous pitcher leaves) are linked to genomic evolution. We found a decaploid karyotype and a clear indication of subgenome dominance. A male-linked and pericentromerically located region on the putative sex chromosome was identified in a recessive subgenome and was found to harbour three transcription factors involved in flower and pollen development, including a likely neofunctionalized LEAFY duplicate. Transcriptomic and syntenic analyses of carnivory-related genes suggested that the paleopolyploidization events seeded genes that subsequently formed tandem clusters in recessive subgenomes with specific expression in the digestive zone of the pitcher, where specialized cells digest prey and absorb derived nutrients. A genome-scale analysis suggested that subgenome dominance likely contributed to evolutionary innovation by permitting recessive subgenomes to diversify functions of novel tissue-specific duplicates. Our results provide insight into how polyploidy can give rise to novel traits in divergent and successful high-ploidy lineages.

Nutritional Composition and Anti-Type 2 Diabetes Mellitus Potential of Femur Bone Extracts from Bovine, Chicken, Sheep, and Goat: Phytochemical and In Vivo Studies.

Nutritional deficits in one's diet have been established as the key risk factor for T2DM in recent years. Nutritional therapy has been demonstrated to be useful in treating T2DM. The current study was carried out to assess the nutritional composition of bovine (12 months), chicken (4 months), sheep (13 months), and goat (9 months) femur bone extracts, as well as their potential therapeutic effects on T2DM regression in a Wistar albino rat model (500 mg/kg b.wt.). The proximate composition of the different extracts, their fatty acid composition, their amino acids, and their mineral contents were identified. In vivo data indicated considerably improved T2DM rats, as seen by lower serum levels of TL, TG, TC, ALT, AST, ALP, bilirubin, creatinine, urea, IL-6, TNF-α, sICAM-1, sVCAM-1, and MDA. Low levels of HDL-C, GSH, and total proteins were restored during this study. Histological investigations of liver and pancreatic tissue revealed that the distribution of collagen fibers was nearly normal. The bovine extract, on the other hand, was the most active, followed by the sheep, goat, and finally chicken extract. This research could result in the creation of a simple, noninvasive, low-cost, and reliable method for T2DM control, paving the way for potential early therapeutic applications in T2DM control.

Ancistrocladinium A Induces Apoptosis in Proteasome Inhibitor-Resistant Multiple Myeloma Cells: A Promising Therapeutic Agent Candidate.

The N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A belongs to a novel class of natural products with potent antiprotozoal activity. Its effects on tumor cells, however, have not yet been explored. We demonstrate the antitumor activity of ancistrocladinium A in multiple myeloma (MM), a yet incurable blood cancer that represents a model disease for adaptation to proteotoxic stress. Viability assays showed a potent apoptosis-inducing effect of ancistrocladinium A in MM cell lines, including those with proteasome inhibitor (PI) resistance, and in primary MM cells, but not in non-malignant blood cells. Concomitant treatment with the PI carfilzomib or the histone deacetylase inhibitor panobinostat strongly enhanced the ancistrocladinium A-induced apoptosis. Mass spectrometry with biotinylated ancistrocladinium A revealed significant enrichment of RNA-splicing-associated proteins. Affected RNA-splicing-associated pathways included genes involved in proteotoxic stress response, such as PSMB5-associated genes and the heat shock proteins HSP90 and HSP70. Furthermore, we found strong induction of ATF4 and the ATM/H2AX pathway, both of which are critically involved in the integrated cellular response following proteotoxic and oxidative stress. Taken together, our data indicate that ancistrocladinium A targets cellular stress regulation in MM and improves the therapeutic response to PIs or overcomes PI resistance, and thus may represent a promising potential therapeutic agent.

Molecular determinants of the response of cancer cells towards geldanamycin and its derivatives.

Geldanamycin is an ansamycin-derivative of a benzoquinone isolated from Streptomyces hygroscopicus. It inhibits tyrosine kinases and heat shock protein 90 (HSP90). Geldanamycin and 11 derivatives were subjected to molecular docking to HSP90, and 17-desmethoxy-17-N,N-dimethylamino-geldanamycin (17-DMAG) was the compound with the highest binding affinity (-7.73 ± 0.12 kcal/mol) and the lowest inhibition constant (2.16 ± 0.49 µM). Therefore, 17-DMAG was selected for further experiments in comparison to geldanamycin. Multidrug resistance (MDR) represents a major problem for successful cancer therapy. We tested geldanamycin and 17-DMAG against various drug-resistant cancer cell lines. Although geldanamycin and 17-DMAG inhibited the proliferation in all cell lines tested, multidrug-resistant P-glycoprotein-overexpressing CEM/ADR5000 cells were cross-resistant, ΔEGFR-overexpressing tumor cells and p53 knockout cells were sensitive to these two compounds. COMPARE and hierarchical cluster analyses were performed, and 60 genes were identified to predict the sensitivity or resistance of 59 NCI tumor cell lines towards geldanamycin and 17-DMAG. The distribution of cell lines according to their mRNA expression profiles indicated sensitivity or resistance to both compounds with statistical significance. Moreover, bioinformatic tools were used to study possible mechanisms of action of geldanamycin and 17-DMAG. Galaxy Cistrome analyses were carried out to predict transcription factor binding motifs in the promoter regions of the candidate genes. Interestingly, the NF-ĸB DNA binding motif (Rel) was identified as the top transcription factor. Furthermore, these 60 genes were subjected to Ingenuity Pathway Analysis (IPA) to study the signaling pathway interactions of these genes. Interestingly, IPA also revealed the NF-ĸB pathway as the top network among these genes. Finally, NF-ĸB reporter assays confirmed the bioinformatic prediction, and both geldanamycin and 17-DMAG significantly inhibited NF-κB activity after exposure for 24 h. In conclusion, geldanamycin and 17-DMAG exhibited cytotoxic activity against different tumor cell lines. Their activity was not restricted to HSP90 but indicated an involvement of the NF-KB pathway.

Pectin Nanoparticle-Loaded Soft Coral Nephthea sp. Extract as In Situ Gel Enhances Chronic Wound Healing: In Vitro, In Vivo, and In Silico Studies.

This study shed light for the first time on the in vivo diabetic wound healing potential activity of natural marine soft coral polymeric nanoparticle in situ gel using an excision wound model. A Nephthea sp. methanol-methylene chloride extract loaded with pectin nanoparticles (LPNs) was created. For the preparation of in situ gel, ion-gelation techniques, the entrapment efficiency, the particle size, the polydispersity index, the zeta potential, the in-vitro drug release, and a transmission electron microscope were used and the best formula was selected. Using (UPLC-Q/TOF-MS), 27 secondary metabolites responsible for extract biological activity were identified. Isolation and identification of arachidic acid, oleic acid, nervonic acid, and bis-(2-ethylhexyl)-phthalate (DEHP) of Nephthea sp. was firstly reported here using NMR and mass spectral analyses. Moreover, LPN in situ gel has the best effects on regulating the proinflammatory cytokines (NF-κB, TNF-α, IL-6, and IL-1ß) that were detected on days 7 and 15. The results were confirmed with an in vitro enzymatic inhibitory effect of the extract against glycogen synthase kinase (GSK-3) and matrix metalloproteinase-1 (MMP-1), with IC50 values of 0.178 ± 0.009 and 0.258 ± 0.011 µg/mL, respectively. The molecular docking study showed a free binding energy of -9.6 kcal/mol for chabrolosteroid E, with the highest binding affinity for the enzyme (GSK-3), while isogosterone B had -7.8 kcal/mol for the enzyme (MMP-1). A pharmacokinetics study for chabrolohydroxybenzoquinone F and isogosterone B was performed, and it predicted the mode of action of wound healing activity.

Abelmoschus eculentus Seed Extract Exhibits In Vitro and In Vivo Anti-Alzheimer's Potential Supported by Metabolomic and Computational Investigation.

Abelmoschus esculentus Linn. (okra, F. Malvaceae) is a fruit widely consumed all over the world. In our study, the anti-Alzheimer's potential of A. esculentus was evaluated. An in vitro DPPH free radical assay on A. esculentus seed's total extract and AChE inhibition potential screening indicated a significant anti-Alzheimer's activity of the extract, which was confirmed through an in vivo study in an aluminum-intoxicated rat model. Additionally, in vivo results demonstrated significant improvement in Alzheimer's rats, which was confirmed by improving T-maze, beam balance tests, lower serum levels of AChE, norepinephrine, glycated end products, IL-6, and MDA. The levels of dopamine, BDNF, GSH, and TAC returned to normal values during the study. Moreover, histological investigations of brain tissue revealed that the destruction in collagen fiber nearly returns back to the normal pattern. Metabolomic analysis of the ethanolic extract of A. esculentus seeds via LC-HR-ESI-MS dereplicated ten compounds. A network pharmacology study displayed the relation between identified compounds and 136 genes, among which 84 genes related to Alzheimer's disorders, and focused on AChE, APP, BACE1, MAPT and TNF genes with interactions to all Alzheimer's disorders. Consequently, the results revealed in our study grant potential dietary elements for the management of Alzheimer's disorders.

Carnivory on demand: phosphorus deficiency induces glandular leaves in the African liana Triphyophyllum peltatum.

Triphyophyllum peltatum, a rare tropical African liana, is unique in its facultative carnivory. The trigger for carnivory is yet unknown, mainly because the plant is difficult to propagate and cultivate. This study aimed at identifying the conditions that result in the formation of carnivorous leaves. In vitro shoots were subjected to abiotic stressors in general and deficiencies of the major nutrients nitrogen, potassium and phosphorus in particular, to trigger carnivorous leaves' development. Adventitious root formation was improved to allow verification of the trigger in glasshouse-grown plants. Among all the stressors tested, only under phosphorus deficiency, the formation of carnivorous leaves was observed. These glandular leaves fully resembled those found under natural growing conditions including the secretion of sticky liquid by mature capture organs. To generate plants for glasshouse experiments, a pulse of 55.4 µM α-naphthaleneacetic acid was essential to achieve 90% in vitro rooting. This plant material facilitated the confirmation of phosphorus starvation to be essential and sufficient for carnivory induction, also under ex vitro conditions. Having established the cultivation of T. peltatum and the induction of carnivory, future gene expression profiles from phosphorus starvation-induced leaves will provide important insight to the molecular mechanism of carnivory on demand.

Ancistrobrevinium A, the first N-methylated, cationic naphthylisoquinoline alkaloid, from the tropical liana Ancistrocladus abbreviatus (Ancistrocladaceae).

Ancistrobrevinium A (1) is the first N-methylated and non-hydrogenated, and thus cationic naphthylisoquinoline alkaloid. It was discovered in the root bark extract of the phytochemically productive West African liana Ancistrocladus abbreviatus (Ancistrocladaceae). Its constitution was elucidated by HR-ESI-MS and 1D and 2D NMR. Due to the steric hindrance in the proximity of the linkage between the naphthalene and isoquinoline parts, the biaryl axis is rotationally hindered. It thus constitutes a stable element of chirality - the only one in the new alkaloid since, different from most other naphthylisoquinoline alkaloids, it has no stereogenic centers. The axial configuration of 1 was assigned by electronic circular dichroism (ECD) investigations, which gave a positive couplet, indicating a 'positive chirality', here corresponding to a P-configuration. Ancistrobrevinium A (1) showed a weak cytotoxic activity against A549 lung cancer cells (IC50 = 50.6 µM).

Jozibrevine D from Ancistrocladus ileboensis, the fifth alkaloid in a series of six possible atropo-diastereomeric naphthylisoquinoline dimers, showing antiparasitic and antileukemic activities.

A new dimeric naphthylisoquinoline alkaloid, jozibrevine D (4e), was isolated from the Central-African liana Ancistrocladus ileboensis. It is a Dioncophyllaceae-type metabolite, being R-configured at C-3 and lacking an oxygen function at C-6 in both isoquinoline moieties. The two identical monomers of jozibrevine D are symmetrically linked via the sterically constrained 3',3''-positions of the naphthalene units so that the central biaryl linkage is rotationally hindered and the alkaloid is, thus, C2-symmetric. With the two outer biaryl bonds being chiral, too, 4e possesses three consecutive stereogenic axes. The absolute stereostructure of the new compound was assigned by 1D and 2D NMR, ruthenium-mediated oxidative degradation, and electronic circular dichroism (ECD) spectroscopy. Jozibrevine D (4e) is the fifth discovered isomer in a series of six possible natural atropo-diastereomeric dimers. It shows potent, and selective, antiprotozoal activity against P. falciparum (IC50 = 0.14 µM), and it also exhibits good cytotoxic activities against drug-sensitive acute lymphoblastic CCRF-CEM leukemia cells (IC50 = 11.47 µM) and their multidrug-resistant CEM/ADR5000 subline (IC50 = 16.61 µM).

Dioncophyllidine E: The first configurationally semi-stable, 7,3'-coupled naphthyldihydroisoquinoline alkaloid, from Ancistrocladus abbreviatus, with antiausterity activity against PANC-1 human pancreatic cancer cells.

The discovery of a new naphthylisoquinoline alkaloid, dioncophyllidine E (4), from the tropical liana Ancistrocladus abbreviatus (Ancistrocladaceae) is described. Due to its rare 7,3'-coupling type, combined with the lack of an oxygen function at C-6, it is configurationally semi-stable at the biaryl axis, and thus occurs as a pair of slowly interconverting atropo-diastereomers, 4a and 4b. Its constitution was assigned mainly by 1D and 2D NMR. The absolute configuration at the stereocenter, C-3, was elucidated by oxidative degradation. The absolute axial configuration of the individual atropo-diastereomers was established by their HPLC resolution, combined with online electronic circular dichroism (ECD) investigations, providing nearly mirror-imaged LC-ECD spectra. These were assigned to the respective atropisomers by ECD comparison with a related, but configurationally stable alkaloid, ancistrocladidine (5). Dioncophyllidine E (4a/4b) exhibits a strong preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrient-deprived conditions, with a PC50 value of 7.4 µM, suggesting its potential as an agent against pancreatic cancer.

Korupensamine A, but not its atropisomer, korupensamine B, inhibits SARS-CoV-2 in vitro by targeting its main protease (Mpro).

By combining docking and molecular dynamics simulations, we explored a library of 65 mostly axially chiral naphthylisoquinoline alkaloids and their analogues, with most different molecular architectures and structural analogues, for their activity against SARS-CoV-2. Although natural biaryls are often regarded without consideration of their axial chirality, they can bind to protein targets in an atroposelective manner. By combining docking results with steered molecular dynamics simulations, we identified one alkaloid, korupensamine A, that atropisomer-specifically inhibited the main protease (Mpro) activity of SARS-CoV-2 significantly in comparison to the reference covalent inhibitor GC376 (IC50 = 2.52 ± 0.14 and 0.88 ± 0.15 µM, respectively) and reduced viral growth by five orders of magnitude in vitro (EC50 = 4.23 ± 1.31 µM). To investigate the binding pathway and mode of interaction of korupensamine A within the active site of the protease, we utilized Gaussian accelerated molecular dynamics simulations, which reproduced the docking pose of korupensamine A inside the active site of the enzyme. The study presents naphthylisoquinoline alkaloids as a new class of potential anti-COVID-19 agents.

Asian Ancistrocladus Lianas as Creative Producers of Naphthylisoquinoline Alkaloids.

This book describes a unique class of secondary metabolites, the mono- and dimeric naphthylisoquinoline alkaloids. They occur in lianas of the paleotropical Ancistrocladaceae and Dioncophyllaceae families, exclusively. Their unprecedented structures include stereogenic centers and rotationally hindered, and thus likewise stereogenic, axes. Extended recent investigations on six Ancistrocladus species from Asia, as reported in this review, shed light on their fascinating phytochemical productivity, with over 100 such intriguing natural products. This high chemodiversity arises from a likewise unique biosynthesis from acetate-malonate units, following a novel polyketidic pathway to plant-derived isoquinoline alkaloids. Some of the compounds show most promising antiparasitic activities. Likewise presented are strategies for the regio- and stereoselective total synthesis of the alkaloids, including the directed construction of the chiral axis.