Greener Strategy for Lupanine Purification from Lupin Bean Wastewaters Using a Molecularly Imprinted Polymer.

Lupanine is an alkaloid used in the pharma industry as a building block or precursor in the synthesis of sparteine and also explored for drug synthesis in the pharma industry as a chiral selector. This alkaloid is found in lupin bean processing wastewaters originated from the debittering process to make these beans edible. In this work, a computational chemistry approach was taken to design molecularly imprinted polymers (MIPs) selecting itaconic acid, a biobased building block, as a functional monomer that can provide higher affinities for lupanine. MIP-1 was prepared using lupanine as the template, itaconic acid as a functional monomer, and ethylene glycol dimethacrylate as a cross-linker by bulk polymerization. Lupanine was concentrated from lupin bean wastewater by nanofiltration, extracted with ethyl acetate, and purified using the synthesized MIP. MIP-1 was able to selectively recognize lupanine and improve the purity of lupanine from 78 to 88%, with 82% recovery of the alkaloid. These results show the potential application of this strategy to render the industrial process more sustainable.

Resolution of alkaloid racemate: a novel microbial approach for the production of enantiopure lupanine via industrial wastewater valorization.

BACKGROUND: Lupanine is a plant toxin contained in the wastewater of lupine bean processing industries, which could be used for semi-synthesis of various novel high added-value compounds. This paper introduces an environmental friendly process for microbial production of enantiopure lupanine. RESULTS: Previously isolated P. putida LPK411, R. rhodochrous LPK211 and Rhodococcus sp. LPK311, holding the capacity to utilize lupanine as single carbon source, were employed as biocatalysts for resolution of racemic lupanine. All strains achieved high enantiomeric excess (ee) of L-(-)-lupanine (> 95%), while with the use of LPK411 53% of the initial racemate content was not removed. LPK411 fed with lupanine enantiomers as single substrates achieved 92% of D-(+)-lupanine biodegradation, whereas L-(-)-lupanine was not metabolized. Monitoring the transcriptional kinetics of the luh gene in cultures supplemented with the racemate as well as each of the enantiomers supported the enantioselectivity of LPK411 for D-(+)-lupanine biotransformation, while (trans)-6-oxooctahydro-1H-quinolizine-3-carboxylic acid was detected as final biodegradation product from D-(+)-lupanine use. Ecotoxicological assessment demonstrated that lupanine enantiomers were less toxic to A. fischeri compared to the racemate exhibiting synergistic interaction. CONCLUSIONS: The biological chiral separation process of lupanine presented here constitutes an eco-friendly and low-cost alternative to widely used chemical methods for chiral separation.

Angustifoline inhibits human colon cancer cell growth by inducing autophagy along with mitochondrial-mediated apoptosis, suppression of cell invasion and migration and stimulating G2/M cell cycle arrest.

PURPOSE: The prime objective of the present study was to investigate the anticancer properties of angustifoline against COLO-205 human colon cancer cells. Its effects on cell autophagy, apoptosis, cell invasion and cell migration, and cell cycle arrest were also evaluated in the current study. METHODS: WST-1 assay was used to study cytotoxic effects of the compound on the cell viability. Effects on apoptosis and cell cycle arrest were evaluated by flow cytometry. In vitro wound healing assay and matrigel assay were carried out to study the effects of angustifoline on cell migration and cell invasion respectively. To confirm autophagy, we evaluated the expression of several autophagy-associated proteins using Western blot assay along with transmission electron microscopy (TEM). RESULTS: The findings indicated that angustifoline induced dose- and time-dependent cytotoxicity in COLO-205 human colon cancer cells along with inhibiting cancer cell colony formation. Angustifoline-treated cells exhibited cell shrinkage along with distortion of the normal cell morphology. Angustifoline-treated cells were also arrested in the G2/M phase of the cell cycle, showing strong dose-dependence. The compound also led to inhibition of cell migration and cell invasion. The results showed that treatment of these cells led to generation of autophagic cell vesicles. Furthermore, it was observed that the expression of Beclin-1 and LC3-II proteins was significantly upregulated in the angustifoline-administered COLO-205 cells. CONCLUSIONS: In brief, the present study hints towards the potent anticancer potential of the natural product angustifoline against COLO-205 human colon cancer cells with in depth mechanistic studies.

17 Oxo Sparteine and Lupanine, Obtained from Cytisus scoparius, Exert a Neuroprotection against Soluble Oligomers of Amyloid-ß Toxicity by Nicotinic Acetylcholine Receptors.

Alzheimer's disease (AD) is a neurodegenerative pathology, which is characterized by progressive and irreversible cognitive impairment. Most of the neuronal perturbations described in AD can be associated with soluble amyloid- ß oligomers (SO-Aß). There is a large amount of evidence demonstrating the neuroprotective effect of Nicotine neurotransmission in AD, mainly through nicotinic acetylcholine receptor (nAChR) activation and antiapoptotic PI3K/Akt/Bcl-2 pathway signaling. Using HPLC and GC/MS, we isolated and characterized two alkaloids obtained from C. scoparius, Lupanine (Lup), and 17- oxo-sparteine (17- ox), and examined their neuroprotective properties in a cellular model of SO-Aß toxicity. Our results showed that Lup and 17- ox (both at 0.03µM) prevented SO-Aß-induced toxicity in PC12 cells (Lup: 64±7%; 17- ox: 57±6%). Similar results were seen in hippocampal neurons where these alkaloids prevented SO-Aß neurotoxicity (Lup: 57±2%; 17- ox: 52±3%) and increased the frequency of spontaneous calcium transients (Lup: 60±4%; 17- Ox: 40±3%), suggesting an enhancing effect on neural network activity and synaptic activity potentiation. All of the neuroprotective effects elicited by both alkaloids were completely blocked by α-bungarotoxin. Additionally, we observed that the presence of both Lup and 17- ox increased Akt phosphorylation levels (52±4% and 35±7%, respectively) in cells treated with SO-Aß (3 h). Taken together, our results suggest that the activation of nAChR by Lup and 17- ox induces neuroprotection in different cellular models, and appears to be an interesting target for the development of new pharmacological tools and strategies against AD.

Discovery of Natural Compounds Promoting Cardiomyocyte Differentiation.

The commitment of pluripotent stem cells to the cardiac lineage has enormous potential in regenerative medicine interventions for several cardiac diseases. Thus, it is necessary to understand and regulate this differentiation process for potential clinical application. In this study, we developed defined conditions with chemical inducers for effective cardiac lineage commitment and elucidated the mechanism for high-efficiency differentiation. First, we designed a robust reporter-based platform to screen chemical inducers of cardiac differentiation in the mouse P19 teratocarcinoma cell line. Using this system, we identified two natural alkaloids, lupinine and ursinoic acid, which enhanced cardiomyocyte differentiation of P19 cells in terms of beating colony numbers with respect to oxytocin, and confirmed their activity in mouse embryonic stem cells. By analyzing the expression of key markers, we found that this enhancement can be attributed to the early and rapid induction of the Wnt signaling pathway. We also found that these natural compounds could not only supersede the action of the Wnt3a ligand but also had a very quick response time, allowing them to act as efficient cardiac mesoderm inducers that subsequently promoted cardiomyocyte differentiation. Thus, this study offers a way to develop chemical-based differentiation strategy for high-efficiency cardiac lineage commitment, which has an advantage over currently available methods with complex medium composition and parameters. Furthermore, it also provides an opportunity to pinpoint the key molecular mechanisms pivotal to the cardiac differentiation process, which are necessary to design an efficient strategy for cardiomyocyte differentiation.

Genome Sequence Analysis of Two Pseudomonas putida Strains to Identify a 17-Hydroxylase Putatively Involved in Sparteine Degradation.

Two strains of Pseudomonas putida, Psp-LUP and Psp-SPAR, capable of growth on the quinolizidine alkaloids, lupanine and sparteine respectively, were studied here. We report the isolation of Psp-SPAR and the complete genome sequencing of both bacteria. Both were confirmed to belong to P. putida, Psp-LUP close to the type isolate of the species (NBRC14164T) and Psp-SPAR close to strains KT2440 and F1. Psp-SPAR did not grow on lupanine but did contain a gene encoding a putative quinolizidine-17-hydroxylase peptide which exhibited high similarity (76%identity) to the lupanine-17-hydroxylase characterised from Psp-LUP.

Bioconversion of alkaloids to high-value chemicals: Comparative analysis of newly isolated lupanine degrading strains.

This work explores the potential for development of a lupanine valorization process evaluating different isolated microorganisms for their capacity to metabolize the alkaloid. Ecotoxicological assessment demonstrated that lupanine is toxic for Vibrio fischeri and Daphnia magna exhibiting EC50 values of 89 mg L-1 and 47 mg L-1 respectively, while acting both as growth inhibitor for a monocotyledonous and as promoter for a dicotyledonous plant. Among the eight aerobic and anaerobic strains isolated and identified Rhodococcus rhodochrous LPK211 achieved 81% removal for 1.5 g L-1 lupanine, while no end-products were detected by NMR constituting a promising microorganism for lupanine biodegradation. Moreover, Rhodococcus ruber LPK111 and Rhodococcus sp. LPK311 exhibited 66% and 71% of removal respectively, including potential formation of lupanine N-oxide. Pseudomonas putida LPK411 reached 80% of lupanine removal and generated three fermentation products potentially comprising 17-oxolupanine and lupanine derivatives with open ring structures enabling the development of alkaloid valorization processes.

In Vivo and In Vitro Activities and ADME-Tox Profile of a Quinolizidine-Modified 4-Aminoquinoline: A Potent Anti-P. falciparum and Anti-P. vivax Blood-Stage Antimalarial.

Natural products are a prolific source for the identification of new biologically active compounds. In the present work, we studied the in vitro and in vivo antimalarial efficacy and ADME-Tox profile of a molecular hybrid (AM1) between 4-aminoquinoline and a quinolizidine moiety derived from lupinine (Lupinus luteus). The aim was to find a compound endowed with the target product profile-1 (TCP-1: molecules that clear asexual blood-stage parasitaemia), proposed by the Medicine for Malaria Venture to accomplish the goal of malaria elimination/eradication. AM1 displayed a very attractive profile in terms of both in vitro and in vivo activity. By using standard in vitro antimalarial assays, AM1 showed low nanomolar inhibitory activity against chloroquine-sensitive and resistant P. falciparum strains (range IC50 16-53 nM), matched with a high potency against P. vivax field isolates (Mean IC50 29 nM). Low toxicity and additivity with artemisinin derivatives were also demonstrated in vitro. High in vivo oral efficacy was observed in both P.berghei and P. yoelii mouse models with IC50 values comparable or better than those of chloroquine. The metabolic stability in different species and the pharmacokinetic profile in the mouse model makes AM1 a compound worth further investigation as a potential novel schizonticidal agent.

Anagyrine desensitization of peripheral nicotinic acetylcholine receptors. A potential biomarker of quinolizidine alkaloid teratogenesis in cattle.

Anagyrine, a teratogenic quinolizidine alkaloid found in Lupinus spp., is proposed to undergo metabolism by pregnant cattle to a piperidine alkaloid which inhibits fetal movement, the putative mechanism behind crooked calf syndrome. The objective of this study was to test the hypothesis that anagyrine but not lupanine or sparteine can directly, without metabolism, desensitize nicotinic acetylcholine receptors (nAChR) in a cell culture model. SH-SY5Y cells expressing autonomic nAChR, and TE-671 cells expressing fetal muscle-type nAChR were exposed to lupine alkaloids or Dimethylphenylpiperazinium (DMPP) in log10 molar increments from 10nM to 100µM and then to a fixed concentration of acetylcholine (ACh) (10µM for SH-SY5Y cells and 1µM for TE-671 cells) and the responses measured with a membrane potential sensing dye to assess nAChR activation and desensitization. The selective ganglionic nAChR agonist DMPP used as a positive control, was a potent activator and desensitizer of nAChR expressed by SH-SY5Y cells. Lupanine was a weak agonist and desensitizer in SH-SY5Y cells and sparteine was without effect. Anagyrine acted as a partial agonist in both cell lines with EC50 values of 4.2 and 231µM in SH-SY5Y and TE-671 cells, respectively. Anagyrine was a desensitizer of nAChR with DC50 values of 6.9 and 139µM in SH-SY5Y and TE-671 cells, respectively. These results confirm the hypothesis that anagyrine is a potent and effective desensitizer of nAChR, and that anagyrine can directly, without metabolism, desensitize nAChR. Moreover, serum anagyrine concentrations may be a potential biomarker for lupine teratogenicity in cattle.

Microclimatic variation in UV perception and related disparity in tropane and quinolizidine alkaloid composition of Atropa acuminata, Lupinus polyphyllus and Hyoscyamus niger.

The aim of current research was to evaluate the physiological adjustment in three medicinal herbs viz., Atropa acuminata, Lupinus polyphyllus and Hyoscyamus niger to the winter period characterised by intense UV flux in Kashmir valley across the North Western Himalaya. Quinolizidine (QA) and tropane alkaloid (TA) concentrations were analysed in these herbs thriving at two different altitudes via GC-MS and correlated by PCA analysis. This study investigated the hypothesis that UV reflectance and absorbance at low temperatures are directly related to disparity in alkaloid accumulation. Among QAs in L. polyphyllus, ammodendrine and lupanine accumulated at higher concentration and exhibited significant variation of 186.36% and 95.91% in ammodendrine and lupanine respectively in both sites. Tetrahydrohombifoline displayed non-significant variation of about 9.60% irrespective of sites. Among tropane alkaloid (TA), hyoscyamine was recorded as the most abundant constituent irrespective of the plant and site while apotropine accumulated in lesser quantity in A. acuminata than H. niger. However, apotropine demonstrated significant variation of 175% among both sites. The final concentration of quinolizidine (QA) and tropane alkaloid (TA) reflects the interplay between reflectance and absorbance of UV radiation response field. These findings suggest that spectral response of UV light contributes directly to alkaloid biosynthesis.

Lupanine Improves Glucose Homeostasis by Influencing KATP Channels and Insulin Gene Expression.

The glucose-lowering effects of lupin seeds involve the combined action of several components. The present study investigates the influence of one of the main quinolizidine alkaloids, lupanine, on pancreatic beta cells and in an animal model of type-2 diabetes mellitus. In vitro studies were performed with insulin-secreting INS-1E cells or islets of C57BL/6 mice. In the in vivo experiments, hyperglycemia was induced in rats by injecting streptozotocin (65 mg/kg body weight). In the presence of 15 mmol/L glucose, insulin secretion was significantly elevated by 0.5 mmol/L lupanine, whereas the alkaloid did not stimulate insulin release with lower glucose concentrations. In islets treated with l-arginine, the potentiating effect of lupanine already occurred at 8 mmol/L glucose. Lupanine increased the expression of the Ins-1 gene. The potentiating effect on secretion was correlated to membrane depolarization and an increase in the frequency of Ca(2+) action potentials. Determination of the current through ATP-dependent K⁺ channels (KATP channels) revealed that lupanine directly inhibited the channel. The effect was dose-dependent but, even with a high lupanine concentration of 1 mmol/L or after a prolonged exposure time (12 h), the KATP channel block was incomplete. Oral administration of lupanine did not induce hypoglycemia. By contrast, lupanine improved glycemic control in response to an oral glucose tolerance test in streptozotocin-diabetic rats. In summary, lupanine acts as a positive modulator of insulin release obviously without a risk for hypoglycemic episodes.

Organocatalytic asymmetric Mannich cyclization of hydroxylactams with acetals: total syntheses of (-)-epilupinine, (-)-tashiromine, and (-)-trachelanthamidine.

An asymmetric, organocatalytic, one-pot Mannich cyclization between a hydroxylactam and acetal is described to provide fused, bicyclic alkaloids bearing a bridgehead N atom. Both aliphatic and aromatic substrates were used in this transformation to furnish chiral pyrrolizidinone, indolizidinone, and quinolizidinone derivatives in up to 89% yield and 97% ee. The total syntheses of (-)-epilupinine, (-)-tashiromine, and (-)-trachelanthamidine also achieved to demonstrate the generality of the process.

An efficient asymmetric synthesis of (-)-lupinine.

The asymmetric synthesis of (-)-lupinine was achieved in 8 steps, 15% overall yield and >99 : 1 dr from commercially available starting materials. The strategy used for the construction of the quinolizidine scaffold involved reaction of an enantiopure tertiary dibenzylamine via two sequential ring-closures which both occurred with concomitant N-debenzylation.

Herbivore defence compounds occur in pollen and reduce bumblebee colony fitness.

Herbivory defence chemicals in plants can affect higher trophic levels such as predators and parasitoids, but the impact on pollinators has been overlooked. We show that defensive plant chemicals can damage pollinator fitness when expressed in pollen. Crop lupins (Lupinus species from Europe and South America) accumulate toxic quinolizidine alkaloids in vegetative tissues, conferring resistance to herbivorous pests such as aphids. We identified the alkaloid lupanine and its derivatives in lupin pollen, and then provided this compound at ecologically-relevant concentrations to queenless microcolonies of bumblebees (Bombus terrestris) in their pollen to determine how foraging on these crops may impact bee colony health and fitness. Fewer males were produced by microcolonies provided with lupanine-treated pollen and they were significantly smaller than controls. This impact on males was not linked to preference as workers willingly fed lupanine-treated pollen to larvae, even though it was deleterious to colony health. Agricultural systems comprising large monocultures of crops bred for herbivore resistance can expose generalist pollinators to deleterious levels of plant compounds, and the broader environmental impacts of crop resistance must thus be considered.

Rotational spectra of bicyclic decanes: the trans conformation of (-)-lupinine.

The conformational and structural properties of the bicyclic quinolizidine alkaloid (-)-lupinine have been investigated in a supersonic jet expansion using microwave spectroscopy. The rotational spectrum is consistent with a single dominant trans conformation within a double-chair skeleton, which is stabilized by more than 10.4 kJ mol(-1) with respect to other conformations. In the isolated conditions of the jet, the hydroxy methyl side chain of the molecule locks in to form an intramolecular O-H···N hydrogen bond to the electron lone pair at the nitrogen atom. Accurate rotational constants, centrifugal distortion corrections, and (14)N nuclear quadrupole coupling parameters are reported and compared to ab initio (MP2) and DFT (M06-2X) calculations. The stability of lupinine is further compared computationally with epilupinine and decaline in order to gauge the influence of intramolecular hydrogen bonding, absent in these molecules.

Amines bearing tertiary substituents by tandem enantioselective carbolithiation-rearrangement of vinylureas.

In the presence of (-)-sparteine or a (+)-sparteine surrogate, organolithiums add to N-alkenyl-N'-arylureas to give benzylic organolithiums in an enantioselective manner. Under the influence of DMPU, these organolithiums undergo rearrangement with migration of the N'-aryl ring from N to C, leading to the urea derivatives of enantiomerically enriched amines bearing tertiary substituents. Basic hydrolysis returns the functionalized amine, providing a new synthetic route to compounds with quaternary stereogenic centers bearing nitrogen.

Synthesis and comparison of antiplasmodial activity of (+), (-) and racemic 7-chloro-4-(N-lupinyl)aminoquinoline.

Recently the N-(-)-lupinyl-derivative of 7-chloro-4-aminoquinoline ((-)-AM-1; 7-chloro-4-{N-[(1S,9aR)(octahydro-2H-quinolizin-1-yl)methyl]amino}quinoline) showed potent in vitro and in vivo activity against both Chloroquine susceptible and resistant strains of Plasmodium falciparum. However, (-)-AM-1 is synthesized starting from (-)-lupinine, an expensive alkaloid isolated from Lupinus luteus whose worldwide production is not sufficient, at present, for large market purposes. To overcome this issue, the corresponding racemic compound, derived from synthetic (±)-lupinine was considered a cheaper alternative for the development of a novel antimalarial agent. Therefore, the racemic and the 7-chloro-4-(N-(+)-lupinyl)aminoquinoline ((±)-AM-1; (+)-AM-1) were synthesized and their in vitro antimalarial activity and cytotoxicity compared with those of (-)-AM-1. The (+)-lupinine required for the synthesis of (+)-AM-1 was obtained through a not previously described lipase catalyzed kinetic resolution of (±)-lupinine. In terms of antimalarial activity, (±)-AM1 and (+)-AM1 demonstrated very good activity in vitro against both CQ-R and CQ-S strains of P. falciparum (range IC(50) 16-35 nM), and low toxicity against human normal cell lines (therapeutic index >1000), comparable with that of (-)-AM1. These results confirm that the racemate (±)-AM1 could be considered as a potential antimalarial agent, ensuring a decrease of costs of synthesis compared to (-)-AM1.

[Reversible lupininin inhibitors of cholinesterases of mammalian blood and of optical ganglia of individuals of the commander squid Berryteuthis magister from different zones of species areal].

Arylsulfoesters and carbonic lupinin esters are studied for the first time as reversible inhibitors of mammalian blood cholinesterases. Studied in detail is sensitivity of cholinesterases to mono- and bislupinin inhibitors in Commander squid individuals from different habitation zones.

[Isomeric derivatives of lupinine and epilupinine--organophosphorus inhibitors of cholinesterases].

The isomeric-structure analysis data of anticholinesterase action of organophosphorous inhibitors with similar structure help in the search of specific effectors and detection of differences in reactivity of various animals' enzymes. This study compared the data of efficacy in respect of 4 mammal and 5 arthropoda cholinesterase preparations for 26 quinolizidine inhibitors, which molecules contain both the isomeric unbranched and branched alkoxyl radicals in the phosphoryl group, and the epimeric lupinine and epilupinine derivatives in the leaving group. The changes in the alkoxyl radical structure of inhibitor molecules act on their efficacy only with respect to the mammal enzymes ("group" inhibitor specificity). The differences between lupinine and epilupinine derivatives were revealed. Highly specific inhibitors of different enzymes were detected among the tested compounds.

[Derivatives of lupinin and epilupinin as ligands of various cholinesterases].

Literature data have been summarized on interaction of cholinesterases of some mammals and arthropods with a group of isomer derivatives of alkaloid lupini and its epimer epilupinin. As substrates of cholinesterases of several mammals there are studied 8 acetates containing in their molecules the chinolysidin bicycle with different structure of N-alkyl radical, which showed certain elements of specificity of action. For 2 isomer esters that are derivatives of the protonated base of the lupinin and epilupinin structures, differences in their substrate characteristics were revealed. The polyenzyme analysis if anticholinesterase efficiency was performed for 30 organophosphorus inhibitors that are dialkoxyphosphorus derivatives of lupinin and epilupinin; as a result, quite a few peculiarities of their action depending on their structure were revealed. Several tested compounds turned out to act as specific inhibitors of cholinesterases of some mammals and arthropods.