Predominant Alkaloid Research Articles

Anticholinergic toxidrome: a rare consequence of lupin ingestion - a case report and literature review

Background: Lupin beans, a yellow legume seed from the Lupinus plant, are commonly used in Southern Europe, Latin America, and the Middle East. Despite their bitter taste, lupin beans are highly nutritious and rich in protein, making them popular snacks. The bitter taste is due to the presence of over 150 quinolizidine alkaloids, with Lupanine being the predominant alkaloid. If not properly debittered before ingestion, lupin toxicity and anticholinergic symptoms can occur. Case Presentation: A 49-year-old healthy Egyptian man presented with symptoms such as dry mouth, unsteadiness, constipation, and urinary retention for 6 hours. After investigation, it was discovered that he had consumed 400 g of bitter lupin beans within a 1-hour period, around 4-6 hours before the symptom onset. The patient was oriented, conscious, and alert, showing no signs of pain or distress. His vital signs were normal, and he had normal cardiovascular, pulmonary, gastrointestinal, and neurological examinations. His eye examination revealed fixed, dilated pupils bilaterally, blurry vision, and normal eye movement bilaterally. Laboratory tests were normal. The patient received Ringer's lactate intravenously and was closely monitored in the emergency department. Over a 3-hour period, all symptoms and signs resolved with fluid management alone, and the patient was discharged home following his improvement. Conclusion: This report highlights the importance of history taking in diagnosing food-related diseases and emphasizes that physicians should consider lupin toxicity as a differential diagnosis in cases presenting with anticholinergic symptoms.

Rhynchophylline inhibits methamphetamine dependence via modulating the miR-181a-5p/GABRA1 axis

Ethnopharmacological relevanceUncaria rhynchophylla (Miq.) Miq. ex Havil. is a plant species that is routinely devoted in traditional Chinese medicine to treat central nervous system disorders. Rhynchophylline (Rhy), a predominant alkaloid isolated from Uncaria rhynchophylla (Miq.) Miq. ex Havil., has been demonstrated to reverse methamphetamine-induced (METH-induced) conditioned place preference (CPP) effects in mice, rats and zebrafish. The precise mechanism is still poorly understood, thus further research is necessary. Aim of studyThis study aimed to investigate the role of miRNAs in the inhibitory effect of Rhy on METH dependence. Materials and methodsA rat CPP paradigm and a PC12 cell addiction model were established. Microarray assays were used to screen and identify the candidate miRNA. Behavioral assessment, real-time PCR, dual-luciferase reporter assay, western blotting, stereotaxic injection of antagomir/agomir and cell transfection experiments were performed to elucidate the effect of the candidate miRNA and intervention mechanism of Rhy on METH dependence. ResultsRhy successfully reversed METH-induced CPP effect and the upregulated miR-181a-5p expression in METH-dependent rat hippocampus and PC12 cells. Moreover, suppression of miR-181a-5p by antagomir 181a reversed METH-induced CPP effect. Meanwhile, overexpression of miR-181a-5p by agomir 181a in combination with low-dose METH (0.5 mg/kg) elicited a significant CPP effect, which was blocked by Rhy through inhibiting miR-181a-5p. Finally, the result demonstrated that miR-181a-5p exerted its regulatory role by targeting γ-aminobutyric acid A receptor α1 (GABRA1) both in vivo and in vitro. ConclusionThis finding reveals that Rhy inhibits METH dependence via modulating the miR-181a-5p/GABRA1 axis, which may be a promising target for treatment of METH dependence.

Metabolite and Molecular Characterization of Mitragyna speciosa Identifies Developmental and Genotypic Effects on Monoterpene Indole and Oxindole Alkaloid Composition.

The monoterpene indole alkaloid (MIA) mitragynine has garnered attention as a potential treatment for pain, opioid use disorder, and opioid withdrawal because of its combined pharmacology at opioid and adrenergic receptors in humans. This alkaloid is unique to Mitragyna speciosa (kratom), which accumulates over 50 MIAs and oxindole alkaloids in its leaves. Quantification of 10 targeted alkaloids from several tissue types and cultivars of M. speciosa revealed that mitragynine accumulation was highest in leaves, followed by stipules and stems, but was absent, along with other alkaloids, in roots. While mitragynine is the predominant alkaloid in mature leaves, juvenile leaves accumulate higher amounts of corynantheidine and speciociliatine. Interestingly, corynantheidine has an inverse relationship with mitragynine accumulation throughout leaf development. Characterization of various cultivars of M. speciosa indicated altered alkaloidal profiles ranging from undetectable to high levels of mitragynine. DNA barcoding and phylogenetic analysis using ribosomal ITS sequences revealed polymorphisms leading M. speciosa cultivars having lower mitragynine content to group with other mitragyna species, suggesting interspecific hybridization events. Root transcriptome analysis of low- and high-mitragynine-producing cultivars indicated significant differences in gene expression and revealed allelic variation, further supporting that hybridization events may have impacted the alkaloid profile of M. speciosa.

In-situ detection and imaging of Areca catechu fruit alkaloids by MALDI-MSI

Arecoline, the predominant alkaloid in the fruit of Areca catechu, is considered the fourth most consumed addictive drug in the world, after nicotine, alcohol, and caffeine. Additionally, the areca nut (AN) is widely used in traditional and herbal medicines. Therefore, performing an exact analysis of alkaloids will provide evidence for more optimal applications of AN. Here, we performed matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and image 10 physiologically active alkaloids in situ (arecoline, arecaidine, caffeine, cotinine, guvacine, guvacoline, hordenine, sophoridine, trigonelline, and vicine) in areca fruits at three different developmental stages. The ion images obtained by MALDI-MSI describe in detail the spatial distributions in different tissues of the fresh fruit of A. catechu, i.e., exocarp, mesocarp, endocarp, episperm, endosperm, and embryos. Liquid chromatography coupled with tandem MS (LC-MS/MS) was also carried out to verify the relative abundance and confirm the quantitative distribution of these alkaloids in Pericarpium Arecae and Semen Arecae. In this study, 3,4-dimethoxycinnamic acid (DMCA) was evaluated as a new matrix for enhanced low-MW compound detection by MALDI-MSI in A. catechu fruit for the first time. New insights into the physiological activities and pharmaceutical properties of areca fruits for further applications can be obtained.

NIC1 cloning and gene editing generates low-nicotine tobacco plants.

Nicotine is the predominant alkaloid in tobacco plants, accounting for ~90% of their total alkaloid content. It is the main addictive substance in cigarettes. Reducing nicotine content in tobacco leaves will aid the development of low nicotine tobacco products. Prior work has shown that manipulation of genes involved in nicotine biosynthesis can achieve this purpose (Hidalgo Martinez et al., 2020). Here, we focused on the long-sought major regulator of nicotine biosynthesis, NIC1 (A).

Analysis of Pyrrolizidine Alkaloids in Queensland Honey: Using Low Temperature Chromatography to Resolve Stereoisomers and Identify Botanical Sources by UHPLC-MS/MS.

Pyrrolizidine alkaloids (PAs) are a diverse group of plant secondary metabolites with known varied toxicity. Consumption of 1,2-unsaturated PAs has been linked to acute and chronic liver damage, carcinogenicity and death, in livestock and humans, making their presence in food of concern to food regulators in Australia and internationally. In this survey, honey samples sourced from markets and shops in Queensland (Australia), were analysed by high-resolution Orbitrap UHPLC-MS/MS for 30 common PAs. Relationships between the occurrence of pyrrolizidine alkaloids and the botanical origin of the honey are essential as pyrrolizidine alkaloid contamination at up to 3300 ng/g were detected. In this study, the predominant alkaloids detected were isomeric PAs, lycopsamine, indicine and intermedine, exhibiting identical MS/MS spectra, along with lesser amounts of each of their N-oxides. Crucially, chromatographic UHPLC conditions were optimised by operation at low temperature (5 °C) to resolve these key isomeric PAs. Such separation of these isomers by UHPLC, enabled the relative proportions of these PAs present in honey to be compared to alkaloid levels in suspect source plants. Overall plant pyrrolizidine alkaloid profiles were compared to those found in honey samples to help identify the most important plants responsible for honey contamination. The native Australian vines of Parsonsia spp. are proposed as a likely contributor to high levels of lycopsamine in many of the honeys surveyed. Botanical origin information such as this, gained via low temperature chromatographic resolution of isomeric PAs, will be very valuable in identifying region of origin for honey samples.

Pyrrolizidine Alkaloids of Blue Heliotrope (Heliotropium amplexicaule) and Their Presence in Australian Honey.

Blue heliotrope (Heliotropium amplexicaule) is an invasive environmental weed that is widely naturalized in eastern Australia and has been implicated as a source of pyrrolizidine alkaloid (PA) poisoning in livestock. Less well-documented is the potential of such carcinogenic alkaloids to contaminate honey from bees foraging on this plant species. In this study, the PA profile of H. amplexicaule plant material, determined by HRAM LC-MS/MS, revealed the presence of nine PAs and PA-N-oxides, including several PAs and PA-N-oxides of the indicine class, which have not previously been reported. The predominant alkaloid, indicine, represents 84% of the reduced PA content, with minor alkaloids identified as intermedine and the newly reported helioamplexine, constituting 7 and 9%, respectively. NMR analysis confirmed the identity of helioamplexine as a previously unreported indicine homologue. This is the first report of the isolation of intermedine, helioamplexine, and 3'-O-angelylindicine from H. amplexicaule. Also described is the identification of N-chloromethyl analogues of the major alkaloids as isolation-derived artifacts from reactions with dichloromethane. Analysis of regional-market honey samples revealed a number of honey samples with PA profiles analogous to that seen in H. amplexicaule, with measured PA contents of up to 2.0 μg of PAs per gram of honey. These results confirm the need for honey producers to be aware of H. amplexicaule as a potential PA source, most particularly in products where honey is sourced from a single location.

Simultaneous Determination of Six Protoalkaloids in Chinese Local Varieties of Loose-Skin Mandarins and Sweet Oranges by Strong Cation Exchange-High Performance Liquid Chromatography

Six protoalkaloids in the fruits of 34 Chinese local loose-skin mandarins and 25 sweet oranges were detected simultaneously by strong cation exchange-high performance liquid chromatography with an ultraviolet detector (SCX-HPLC-UV). Linear regression analysis showed satisfactory linearity for all the analytes with the correlation coefficients (R2) higher than 0.9998. The satisfactory recoveries of the six protoalkaloids in pulps at two spiked levels ranged from 82.8 to 93.4% (RSDs, 1.5–6.1%) and those of synephrine in peels ranged from 89.3 to 91.9% (RSDs, 4.8–5.3%). The limits of quantification (LOQs) were 0.2 mg kg−1. The proposed method was used to determinate the protoalkaloids in loose-skin mandarins and sweet oranges to evaluate its feasibility and accuracy. The major alkaloid in the tested samples was synephrine, and the predominant alkaloid was N-methyltyramine, followed by octopamine. The variations in alkaloid components and contents were both largely the same for the tested varieties, verifying that alkaloids in plants are genetically controlled.

In vitro anticholinesterase, antimonoamine oxidase and antioxidant properties of alkaloid extracts from kola nuts (Cola acuminata and Cola nitida).

Background The development of cholinesterase (ChE) and monoamine oxidase (MAO) inhibitors for management of neurodegenerative diseases such as Alzheimer's disease (AD) has come with their undesirable side effects. Hence, research for potent but natural ChE and MAO inhibitors with little or no side effects is essential. This study investigated the potentials of alkaloid extracts from two Cola species as nutraceuticals for prevention and management of AD. Methods Alkaloid extracts were obtained from two Cola species (Cola nitida [KN] and Cola acuminata [KA]) by solvent extraction method. The extracts were characterized for their alkaloid contents using gas chromatography (GC). The effects of the extracts on ChE and MAO activities were investigated in vitro. Also, the extracts' ability to inhibit Fe2+-induced lipid peroxidation in rat brain homogenate, scavenge DPPH and OH radicals, as well as chelate Fe2+ were determined. Results GC characterization revealed the presence of augustamine and undulatine as the predominant alkaloids in the extracts. There was no significant (P > 0.05) difference in the inhibitory effects of the extracts on ChE activities. However, KA extract exhibited significantly higher (P < 0.05) MAO inhibitory effect than KN. Also, KA extract inhibited Fe2+- induced malondialdehyde (MDA) production in rat brain homogenate more significantly than KN, while there was no significant difference in DPPH and OH radicals scavenging, as well as Fe2+-chelating abilities of the extracts. Conclusions Our findings revealed that KN and KA alkaloid extracts exhibited significant effect in vitro on biological pathways that may contribute to neuroprotection for the management of neurodegenerative diseases.

Quaternary alkaloids in Chelidonium majus in vitro cultures

Chelidonium majus L. is a medicinal plant containing several alkaloids, such as chelidonine, chelerythrine, sanguinarine, protopine, and coptisine that are stored and secreted from laticifers—highly specialized elongated cells of aerial and terrestrial parts of the plant. For use as a drug, several cultivars of this specialized crop are cultivated but the content and composition of bioactive substances is variable. Based on the observation of a characteristic orange-yellow color of latex in dedifferentiated tissue of in vitro callus cultures we investigated the alkaloid composition and the anatomy of callus and organ cultures of C. majus maintained on media enriched in plant growth regulators and illuminated with white or photosynthetically active light. Alkaloid composition was analyzed using DAD-HPLC, whereas tissue and organ anatomy was studied using light and scanning electron microscopy.The fully developed organs and rhizogenic callus compared to the undifferentiated cells, produced larger proportion of alkaloids, and complex alkaloid composition. In roots, sanguinarine was the predominant alkaloid exceeding 4 mg/g dry mass. In microshoots, coptisine was the major compound, with the maximum content exceeding 14 mg/g, depending on the medium composition. In callus cultures, either protopine or sanguinarine predominated on different media and under different illumination, with maximum content over 13 mg/g dry mass of protopine reached under PAR (Photosynthetically Active Radiation), and sanguinarine over 0.9 mg/g. The presence of laticifers was confirmed with microscopy and the scanning electron microscopy revealed an abundant extracellular matrix in the callus tissues.These results suggest the high biosynthetic potential of in vitro grown organs and dedifferentiated callus tissue that are able to produce significant amounts of pharmacologically relevant alkaloids from C. majus in various proportions that depend on the culture conditions such as supplementation with growth substances and sugars as well as on the illumination with light of different spectrum.

Proportions of predominant Ergot alkaloids (Claviceps purpurea) detected in Western Canadian grains from 2014 to 2016

Ergot alkaloids, produced by the fungusClaviceps purpurea, are contaminants of cereal crops. Depending on various factors, the relative composition of individual ergot alkaloids can differ among samples. The objective was to determine if the percentage of individual ergot alkaloids were similar across different cereal grains (barley n=39, rye n=7, triticale n=9, wheat n=94) collected in Western Canada over different years. Ergocristine was the predominant alkaloid accounting for half of the total alkaloids in all grain types. This study documented that barley, rye, triticale and wheat collected across Western Canada had similar percentages of ergocornine (6±1%,P=0.201), ergocristine (48±2%,P=0.939), ergocryptine (17±2%,P=0.302) and ergosine (5±0.5%,P=0.239). There were differences between grain types for ergometrine (P=0.027) and ergotamine (P=0.011), which ranged between 6 to 13% and 11 to 24%, respectively, of the total alkaloid content in different cereals. Both barley and wheat alkaloid percentages were similar between 2015 and 2016; ergocornine (7±1%,P=0.969), ergocristine (47±2%,P=0.680), ergocryptine (18±2%,P=0.572), ergometrine (8±1%,P=0.080), ergosine (15±1%,P=0.119) and ergotamine (P=0.189). The ergocornine percentage was higher in wheat (P=0.017) as compared to barley for 2015/2016 samples. Ergometrine was higher in barley (P=0.002) as compared to wheat for 2015/2016 samples. While two of the alkaloid proportions varied statistically, overall proportions of the six ergot alkaloids were comparable among the four grain types collected across Western Canada. If proportions of ergot alkaloids are similar across a region, then it may be deemed acceptable to recommend a maximum total ergot alkaloid concentration for that region. However, areas that exhibit variation among the ergot alkaloid proportions, individual ergot alkaloid guidelines based on a toxic equivalence factor, may be more appropriate. In contrast, since major differences were not seen between years or grain type, from a producer perspective there may be limited biological/toxicological significance for individual alkaloid guidelines.

Study of salivary arecoline in areca nut chewers.

Aims:Arecoline, a predominant alkaloid present in arecanut, has been implicated in the pathogenesis of several oral diseases because of its mutagenic and carcinogenic potential. The response of cultured cells to arecoline is highly dependent on its concentration; arecoline stimulates cultured cells above 0.1 μg/ml and is cytotoxic above 10 μg/ ml. Although this alkaloid seems important for areca nut induced oral diseases and carcinogenesis, little is known of the levels achieved before, during and after chewing. Also, it is prudent to understand its effects in arecanut chewers for a comprehensive understanding of its pathogenesis. Accordingly, the present study quantified the salivary arecoline levels in arecanut chewers.Materials and Methods:The study participants were divided into Study Group A & B and Control Group C; unstimulated whole saliva was collected by spitting method for a period of 5 min. Then, participants in Group A and C chewed 0.5 g of areca nut without any other additives while in Group B were asked to chew 0.5 g of inert rubber base impression material. Stimulated whole saliva from all three groups was collected into graduated tubes during chewing at time intervals of 1, 3, 5, 10, 15, 20 and 25 min. Then, all participants were asked to remove nut particles or inert rubber base material from the mouth, and saliva samples were collected further up to 20 min, changing tubes at 5 min interval. Salivary arecoline was quantitated by HPLC-MS. The tabulation and descriptive statistics of the study were carried out.Results:In the present study, baseline levels of arecoline were zero in all three groups, whereas mean salivary arecoline levels during chewing were 76.93 ng/ml, 129.83 ng/ml and 64.83 ng/ml and after chewing were 196.17 ng/ml, 321.12 ng/ml and 43.75 ng/ml in Groups A, B and Control respectively, which were significantly higher than reported threshold levels.Conclusions:The data from this study reveals that a significant amount of arecoline would be trapped in oral cavity, or being re-circulated between blood and saliva might have resulted in surprisingly high levels of arecoline even 10 mins after chewing in both groups after which the levels started declining. The higher levels of salivary arecoline achieved during and after chewing are enough to cause cytotoxic and genotoxic effects on oral tissues over a period of time in chronic chewers. The great differences in salivary arecoline levels achieved during chewing, may contribute to the variable response to areca nut seen in communities where this habit is widespread. Areca nut users have persistent background salivary arecoline levels long after chewing, whereas concentrations achieved are highly variable and consistent with a role in oral pre-malignancy and malignancy..

Use of isoquinoline alkaloids as markers for identification of honey and pollen from Macleaya cordata (Willd.) R. Br

Macleaya cordata (Willd.) R. Br. (McRB) is utilized in traditional medicine and is mainly distributed in North America, Europe, Japan, and China. McRB honey known as “mad honey” produced by honey bees from the nectar of McRB contains isoquinoline alkaloids that give rise to potential consumer risk. In this study, a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) extraction procedure followed by ultra-high performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UHPLC–MS/MS) was developed and optimized for the determination of seven isoquinoline alkaloids in McRB bee pollen and honey. The results revealed the presence of seven alkaloids in McRB bee pollen, four of which were also detected in McRB honey. Protopine and allocryptopine were the two predominant alkaloids, with concentrations of 0.17–0.66 mg/kg and 0.068–0.19 mg/kg in McRB honey and 1.25*10^3–3.07*10^3 mg/kg and 1.12*10^3–2.52*10^3 mg/kg in McRB bee pollen, respectively. None of the seven alkaloids were detected in commercial honey (n = 130) or pollen samples (n = 30). This study shows that protopine and allocryptopine could serve as potential markers of honey and pollen specifically from McRB.

Phytochemical Profile and Antibacterial Activity of Retama raetam and R. sphaerocarpa cladodes from Algeria

Retama raetam( RR) and R. sphaerocarpa( RS) are shrubs growing in Algeria desert areas, where are commonly used as healing remedies because of their antiseptic, antipyretic and anti-diarrheal properties. Phytochemical studies have shown that these species are very rich in flavonoids (isoflavones) and alkaloids (quinolizidine and bipiperidyl). The aim of this study was to compare the chemical composition of both Retama species by GC/MS and LC/MS and to determinate their antimicrobial activity of two Retama species growing naturally in Algeria. Ten alkaloids and seven flavonoids were identified in cladodes of RR and RS. The quantitative analysis showed that the most abundant flavonoid of both the aqueous extract from RR and RS was the isoflavone genistein (610.0±2.8 and 408.0±14.1 mg/100 g respectively), whereas sparteine was the predominant alkaloid in RR and retamine in RS. The antibacterial activity of Retama extracts against standard strains was performed by minimum inhibitory concentration (MIC), and by the disc diffusion method (expressed by inhibition zone, IZ). Both Retama species showed the best activity against Staphylococcus aureus and methicillin-resistant S. aureus(MRSA), being RS aqueous extract more active than RR aqueous extract, with MIC 125 μg/mL and bactericidal activity against both strains.

Liquid chromatography-tandem mass spectrometric assay for determination of unstable arecoline in rat plasma and its application.

Arecoline, a predominant alkaloid in areca nut, shows several pharmacological and toxicological effects. In present study, a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for quantification of arecoline in rat plasma. Importantly, our group found that arecoline was highly unstable in rat plasma samples, which brought challenges to accurately quantify in vivo. The hydrolysis of ester moiety of arecoline by carboxylesterases was responsible for its instability, and arecoline was hydrolyzed to arecaidine. The degradation of arecoline was completely inhibited using 5% formic acid as a stabilizer, which was immediately added to freshly collected rat plasma samples. EDTA was adopted as the anticoagulant to also reduce the degradation during blood collection and plasma separation owing to its anti-esterase effect. The plasma sample was separated by a C18 analytical column with a mobile phase of 0.1% formic acid and methanol (95:5v/v) at an isocratic flow rate of 300μL/min. The analyte was monitored on a tandem mass spectrometer using the multiple reaction monitoring scan in a positive electrospray ionization mode. The method exhibited high sensitivity and a good linearity rang of 1-1000ng/mL. The developed analytical method was employed in a pilot pharmacokinetic study of arecoline in rats. Arecoline was rapidly eliminated within 45min in rats after oral treatment of 150mg/kg arecoline.

A ZnS-Nanoparticle-Label-Based Electrochemical Codeine Sensor

Codeine (3-methylmorphine) is an opiate that is widely used to treat mild or moderate pain and cough suppression. It is the second predominant alkaloid in opium with a mild sedative effect. In the present study, we describe an electrochemical sensor for codeine detection by using the DNA aptamers against codeine. In the sensing protocol, a dually-labeled DNA aptamer probe was designed to be labeled at one end with HS, and at its another end with dabcyl as an electrochemical tag to produce electrochemical signal via recognization occurrence. One special electrochemical marker was prepared by modifying ZnS nanoparticle with-cyclodextrins (ab. ZnS-CDs), which employed as electrochemical signal provider and would conjunct with the codeine probe modified electrode through the host–guest recognition of CDs to dabcyl. With codeine adding, aptamer folding allows the ZnS-CDs into soultion that caused an increase of current signal. This sensor has the ability to detect 37pM codeine. Our study demonstrates that the biosensor has good specificity and stability. It can be used to detect codeine.

Genomic Insights into the Evolution of the Nicotine Biosynthesis Pathway in Tobacco.

In tobacco (Nicotiana tabacum), nicotine is the predominant alkaloid. It is produced in the roots and accumulated mainly in the leaves. Jasmonates play a central signaling role in damage-induced nicotine formation. The genome sequence of tobacco provides us an almost complete inventory of structural and regulatory genes involved in nicotine pathway. Phylogenetic and expression analyses revealed a series of structural genes of the nicotine pathway, forming a regulon, under the control of jasmonate-responsive ETHYLENE RESPONSE FACTOR (ERF) transcription factors. The duplication of NAD and polyamine metabolic pathways and the subsequent recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon were suggested to be the drivers for pyridine and pyrrolidine ring formation steps early in the pathway. Transcriptional regulation by ERF and cooperatively acting MYC2 transcription factors are corroborated by the frequent occurrence of cognate cis-regulatory elements of the factors in the promoter regions of the downstream structural genes. The allotetraploid tobacco has homologous clusters of ERF genes on different chromosomes, which are possibly derived from two ancestral diploids and include either nicotine-controlling ERF189 or ERF199 A large chromosomal deletion was found within one allele of the nicotine-controlling NICOTINE2 locus, which is part of one of the ERF gene clusters, and which has been used to breed tobacco cultivars with a low-nicotine content.

Digital Gene Expression Analysis Provides Insight into the Transcript Profile of the Genes Involved in Aporphine Alkaloid Biosynthesis in Lotus (Nelumbo nucifera).

The predominant alkaloids in lotus leaves are aporphine alkaloids. These are the most important active components and have many pharmacological properties, but little is known about their biosynthesis. We used digital gene expression (DGE) technology to identify differentially-expressed genes (DEGs) between two lotus cultivars with different alkaloid contents at four leaf development stages. We also predicted potential genes involved in aporphine alkaloid biosynthesis by weighted gene co-expression network analysis (WGCNA). Approximately 335 billion nucleotides were generated; and 94% of which were aligned against the reference genome. Of 22 thousand expressed genes, 19,000 were differentially expressed between the two cultivars at the four stages. Gene Ontology (GO) enrichment analysis revealed that catalytic activity and oxidoreductase activity were enriched significantly in most pairwise comparisons. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, dozens of DEGs were assigned to the categories of biosynthesis of secondary metabolites, isoquinoline alkaloid biosynthesis, and flavonoid biosynthesis. The genes encoding norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), N-methylcoclaurine 3′-hydroxylase (NMCH), and 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) in the common pathways of benzylisoquinoline alkaloid biosynthesis and the ones encoding corytuberine synthase (CTS) in aporphine alkaloid biosynthetic pathway, which have been characterized in other plants, were identified in lotus. These genes had positive effects on alkaloid content, albeit with phenotypic lag. The WGCNA of DEGs revealed that one network module was associated with the dynamic change of alkaloid content. Eleven genes encoding proteins with methyltransferase, oxidoreductase and CYP450 activities were identified. These were surmised to be genes involved in aporphine alkaloid biosynthesis. This transcriptomic database provides new directions for future studies on clarifying the aporphine alkaloid pathway.

Determination of mycotoxins, alkaloids, phytochemicals, antioxidants and cytotoxicity in Asiatic ginseng (Ashwagandha, Dong quai, Panax ginseng)

Mycotoxins and selected hazardous alkaloids in the medicinal plants (Panax ginseng, Angelica sinensis, and Withania somnifera) and dietary supplements were determined. Purine alkaloids were found in majority of samples; however, isoquinoline alkaloids were less abundant than indole. The predominant alkaloids appear to be caffeine (purine group), harman (indole group) and berberine (isoquinoline). Examined medicinal plants and dietary supplements were contaminated by mycotoxins (especially ochratoxin A 1.72–5.83 µg kg−1), and many species of mold (e.g. Cladosporium, Eurotium, Aspergillus, Rhizopus, Penicillium). MTT cytotoxicity tests revealed that plant and supplements extracts exhibited medium or high cytotoxicity (only Dong quai—low). Moreover, antioxidant activity, total phenolics content and selected phytochemicals were analyzed by spectrophotometric and chromatographic methods. Quercetin and rutin were predominant flavonols (1.94-9.51 and 2.20–7.28 mg 100 g−1, respectively). Analysis of phenolic acids revealed—gallic acid, as the most abundant, except Panax ginseng, where ferulic acid was prevailing. The results were analyzed by chemometric methods (cluster analysis, ANOVA).

The effects of selected factors on measured ergot alkaloid content inClaviceps purpurea-infected hexaploid and durum wheat

Four wheat genotypes, including the ergot-susceptible durum ‘AC Avonlea’ and hard red spring wheat ‘AC Cadillac’, as well as the resistant durum wheat line 9260B-173A and the hard red spring wheat line ‘Kenya Farmer’ wereinoculated with differentClaviceps purpurea isolates. Honeydew and sclerotia were collected and analysed for 10 ergot alkaloids. Total concentrations of the 10 ergot alkaloids ranged from 16 µg/kg in honeydew to 1,798 mg/kg insclerotia. Ergonovine and ergosine were the predominant alkaloids in honeydew obtained from plants inoculated with various isolates, whereas ergocristine and ergocryptine were the main alkaloids observed in sclerotia. Bothhost plant andC. purpurea isolate were significant factors affecting total ergot alkaloid concentrations in sclerotia. Irrespective of host plant line, all mean total ergot alkaloid concentrations were higher in sclerotia produced from the EI-2 isolate (695-1,010 mg/kg), as compared to EI-4 (255-594 mg/kg). The mass of total ergot alkaloids was alsopositively correlated with the mass of individual sclerotia produced from these twoC. purpurea isolates, with the slope of the regression higher for the EI-2 isolate. The total ergot alkaloid concentrations in sclerotia from various plants inoculated with the sameC. purpurea isolate differed; however, the resistance of host plant line did notappear to be consistent with ergot alkaloid content in sclerotia. Concentrations of total ergot alkaloids were highestand lowest in sclerotia from the two lines that are both classified as ‘resistant’, suggesting that the mechanism ofresistance for these lines is not restriction on the production of ergot alkaloids in sclerotia.