Rapid selective screening and determination of ephedrine alkaloids using GC-MS footnote mark

Ephedra (ma huang) has been widely used as an herb or herbal extract in both traditional Chinese medicine and Western world dietary supplements. The effects of Ephedra have been attributed to a series of six ephedrine alkaloids including ephedrine and pseudoephedrine. A GC-MS method for the ephedrine alkaloids is described which couples ammoniacal chloroform as the extraction solvent with a two-stage derivatisation scheme. This scheme produces the O-trimethylsilyl, N-trifluoracetyl derivatives (O-TMS, N-TFA) for the primary and secondary amine alkaloids, and the O-TMS derivatives for the tertiary amine alkaloids. Relatively clean extracts are obtained from complex matrices, and the six ephedrine alkaloids are effectively separated and identified. This approach was also evaluated for quantitative analysis, and was shown to provide quantitative results for ephedrine and pseudoephedrine, and good estimates for the four minor alkaloids. Figures of merit are presented for linearity, detection limits, precision and accuracy. We have applied this approach to the rapid screening and profiling of the ephedrine alkaloids in whole Ephedra plants, liquid plant extracts, dried powder plant extracts and a variety of Ephedra-containing dietary supplements.     

麻黄资源的利用与研究开发进展

本文介绍了麻黄资源的现状、麻黄碱的特性以及代谢途径 ,评述了有机合成、植物提取、组织培养以及微生物发酵半合成等生产麻黄碱方法及存在的问题 ,详细讨论了近年来麻黄组织培养的进展及其影响因素。强调生物技术应是解决麻黄碱生产中资源枯竭和保护环境的有效手段。     

Identification of Erythroxylum taxa by AFLP DNA analysis

Erythroxylum coca, indigenous to the Andean region of South America, is grown historically as a source of homeopathic medicine. However, in the last century, cultivation of E. coca and several closely-related species for the production of illicit cocaine has become a major global problem. Two subspecies, E. coca var. coca and E. coca var. ipadu, are almost indistinguishable phenotypically; a related cocaine-bearing species also has two subspecies (E. novogranatense var. novogranatense and E. novogranatense var. truxillense) that are phenotypically similar, but morphologically distinguishable. The purpose of this research was to discover unique AFLP DNA patterns ("genetic fingerprinting") that characterize the four taxa and then, if successful, to evaluate this approach for positive identification of the various species of coca. Of seven different AFLP primer pairs tested, a combination of five proved optimal in differentiating the four taxa as well as a non-cocaine-bearing species, E. aerolatum. This method of DNA fragment separation was selective, and faster, for coca identification, compared with analyses based on flavonoid chemotaxonomy. Using the 5-primer AFLP approach, 132 known and unknown coca leaf accessions were evaluated. Of these, 38 were collected in 1997-2001 from illicit coca fields in Colombia, and all were genetically differentiated from coca originating in Peru and Bolivia. Based on the DNA profiling, we believe that the Colombian coca now represents a hybridization of E. coca var. ipadu. Geographical profiling within Colombia also seems feasible as new coca production areas are developed or new types of coca are introduced within traditional growing areas.     

麻黄资源的利用与研究开发进展

本文介绍了麻黄资源的现状、麻黄碱的特性以及代谢途径,评述了有机合成、植 物提取、组织培养以及微生物发酵半合成等生产麻黄碱方法及存在的问题,详细讨论了近年来麻黄组织培养的进展及其影响因素。强调生物技术应是解决麻黄碱生产中资源枯竭和保护环境的有效手段。     

Chemotaxonomy of Portuguese Ulex: quinolizidine alkaloids as taxonomical markers

Six species of Portuguese Ulex L. in a total of nineteen populations were studied by GC-EIMS as to their content in quinolizidine alkaloids. Sparteine, beta-isosparteine, jussiaeiine A, N-methylcytisine, cytisine, 5,6-dehydrolupanine, rhombifoline, lupanine, jussiaeiine B, N-formylcytisine, N-acetylcytisine, anagyrine, jussiaeiine C, jussiaeiine D, pohakuline, baptifoline, and epibaptifoline were detected. Analysis of the chromatograms showed that the chemical profile of all species was mainly composed of N-methylcytisine, cytisine, anagyrine, and jussiaeiines A, B, C and D. Therefore a quantification study of these alkaloids in all the populations studied was done by GC. These data were then submitted to cluster analysis and principal component analysis, which allowed the definition of five chemotypes and the recognition of hybrids. N-methylcytisine, cytisine, and jussiaeiines A, C and D are recognized as markers of this genus in Portugal.     

毛竹浸提液对苦槠幼苗生长的化感效应

为探讨毛竹(Phyllostachys edulis)扩张过程中潜在的化感作用,选择苦槠(Castanopsis sclerophylla(Lindl)Schott)为研究对象。采用水浸提的方法,用毛竹茎叶、枯落物和土壤3部分浸提液浇灌苦槠幼苗,以蒸馏水处理作为对照,对比分析质量浓度分别为0.1、0.05、0.02 g/mL的3个浓度梯度浸提液处理下苦槠幼苗生长指标及各项光合生理指标的差异。结果表明,毛竹浸提液对苦槠幼苗苗高、地径和叶绿素相对含量的影响大体上呈现高浓度抑制低浓度促进的双重浓度效应。不同来源毛竹浸提液的化感效应不尽相同,土壤浸提液对苦槠幼苗生长和光合生理均呈现抑制作用,而茎叶、枯落物浸提液低浓度时为促进作用。毛竹潜在的化感作用,在其扩张过程中可能会干扰森林主要树种更新,从而对森林群落产生威胁。     

Bat frugivory in two subtropical rain forests of Northern Argentina: Testing hypotheses of fruit selection in the Neotropics

Phyllostomid bats are prominent components of mammalian assemblages in the Neotropics. With many species specialized in frugivory, phyllostomids represent major partners of fleshy-fruited plants in the mutualism of seed dispersal. Here we present dietary data from two subtropical rainforests of Argentina, where fruit diversity is low and thus offer unique opportunities to test hypotheses of diet selection originally proposed for species-rich tropical assemblages. Particularly, we tested whether frugivorous phyllostomids exhibit pronounced dietary specialization in core plant taxa where fruit offer is greatly reduced as compared to tropical rainforests. We analyzed dietary overlap and niche breadth of subtropical frugivorous bats on the basis of >1000 dietary records plus >500 samples from a previous study in the region. We show that in the subtropics, frugivores from different genera remain faithful to their respective core plant taxa with few exceptions, rather than shifting toward alternative fruit resources available in the study sites. This supports predictions of specialization, which is confirmed to have a deep historical origin. The response of phyllostomid ensembles to restricted fruit diversity is at the level of species composition: absence of species for which preferred fruits do not occur in the sites. Taken together, these data lend strong support to hypotheses that explain coexistence of frugivorous phyllostomids on the basis of dietary specialization on core plant taxa with chiropterochorous fruits.     

Acetylcholinesterase inhibitory activity of some Amaryllidaceae alkaloids and Narcissus extracts

Amaryllidaceous plants produce pharmacologically active alkaloids, galanthamine being the most interesting for its use in the treatment of Alzheimer's disease as a cholinesterase inhibitor. The aim of this work was to test 23 pure Amaryllidaceae alkaloids and 26 extracts from different species of the genus Narcissus for their acetylcholinesterase inhibitory activity using galanthamine as a reference. Only seven alkaloids, belonging to the galanthamine and lycorine skeleton types, exhibited such an effect, sanguinine being the most active, even more than galanthamine. All the extracts with the highest acetylcholinesterase inhibitory activity contained galanthamine except that of N. assoanus, a lycorine type alkaloid-bearing species.     

A novel plant-fungal mutualism associated with fire

Bromus tectorum, or cheatgrass, is native to Eurasia and widely invasive in western North America. By late spring, this annual plant has dispersed its seed and died; its aboveground biomass then becomes fine fuel that burns as frequently as once every 3-5 y in its invaded range. Cheatgrass has proven to be better adapted to fire there than many competing plants, but the contribution of its fungal symbionts to this adaptation had not previously been studied. In sampling cheatgrass endophytes, many fire-associated fungi were found, including Morchella in three western states (New Mexico, Idaho, and Washington). In greenhouse experiments, a New Mexico isolate of Morchella increased both the biomass and fecundity of its local cheatgrass population, thus simultaneously increasing both the probability of fire and survival of that event, via more fuel and a greater, belowground seed bank, respectively. Re-isolation efforts proved that Morchella could infect cheatgrass roots in a non-mycorrhizal manner and then grow up into aboveground tissues. The same Morchella isolate also increased survival of seed exposed to heat typical of that which develops in the seed bank during a cheatgrass fire. Phylogenetic analysis of Eurasian and North American Morchella revealed that this fire-associated mutualism was evolutionarily novel, in that cheatgrass isolates belonged to two phylogenetically distinct species, or phylotypes, designated Mel-6 and Mel-12 whose evolutionary origin appears to be within western North America. Mutualisms with fire-associated fungi may be contributing to the cheatgrass invasion of western North America.     

Discovery of skin alkaloids in a miniaturized eleutherodactylid frog from Cuba

Four phylogenetically independent lineages of frogs are currently known to sequester lipid-soluble skin alkaloids for which a dietary source has been demonstrated. We report here a remarkable fifth such instance, in Eleutherodactylus iberia and Eleutherodactylus orientalis, two species of miniaturized frogs of the family Eleutherodactylidae from Cuba. Six pumiliotoxins and two indolizidines were found in E. iberia, one of the smallest frogs in the world and characterized by a contrasting colour pattern for which we hypothesize an aposematic function. Analyses of stomach content indicated a numerical prevalence of mites with an important proportion of oribatids-a group of arthropods known to contain one of the pumiliotoxins detected in E. iberia. This suggests that miniaturization and specialization to small prey may have favoured the acquisition of dietary skin alkaloids in these amphibians.     

Dopamine-iridoid alkaloids in Carapichea affinis (= Psychotria borucana) confirm close relationship to the vomiting root Ipecac

Broad-based HPLC-UV comparison of 57 methanolic crude extracts of the Psychotria complex (Rubiaceae), representing about 20 different species collected in Costa Rica, exhibited a clear chemical segregation of Psychotria borucana. In contrast to the more widespread tryptamine-iridoid alkaloids it deviated by an accumulation of dopamine-iridoid alkaloids. Similar to the South American vomiting root Ipecac, Psychotria ipecacuanha (= Cephaelis ipecacuanha), cephaeline and emetine were found together with four related glycosides characterized by various N-acylations. Two glycosides were identified as the known N-acetates ipecoside and 6-O-methylipecoside, whereas the two others represent corresponding N-O-methylcaffeate derivatives from which borucoside was described as a new compound. Structure elucidation of all compounds was carried out by NMR- and MS-analyses supported by CSEARCH-NMR-database system. In addition to morphological characters and analyses of nucleotide sequence variation the chemical profile of P. borucana supported a recent taxonomic rearrangement where it has been grouped together with P. ipecacuanha in a separate genus Carapichea.     

Diversity,host associations,and phylogeography of temperate aurofusarin-producing Hypomyces/Cladobotryum including causal agents of cobweb disease of cultivated mushrooms

Temperate species of Hypomyces and Cladobotryum that produce the red pigment aurofusarin are common on agaricoid and polyporoid basidiomata of species from five orders of Agaricomycetes. Several cause cobweb disease of commercially cultivated mushrooms resulting in serious losses. We sequenced rpb1, rpb2, tef1, and FG1093 regions in 90 wild strains and 30 strains from mushroom farms, isolated from Europe, North America, Africa, Asia, Australia, and New Zealand. Multigene analyses support the distinctness of five species but reveal Hypomyces rosellus to be paraphyletic, comprising several cryptic lineages. Hypomyces rosellus s. str. is characterised by wide dispersal and gene flow across Eurasia but does not occur in North America. Instead, the lineages from the West and the East Coast appear distinct, having given rise to species inhabiting the Southern Hemisphere. Our results reveal wide misuse of the name H. rosellus, especially for cobweb isolates. The majority of these belong to Hypomyces odoratus, including a weakly supported group of fungicide-resistant strains from Europe and North America sharing identical sequence data. New collections are presented for Cladobotryum rubrobrunnescens and Cladobotryum tenue as well as Cladobotryum multiseptatum and Hypomyces dactylarioides, all previously known only from their type material. The former species pair occurs in Europe and the latter in Australia and New Zealand. Separate lineages appear to be maintained by geographic isolation in North America and temperate Australasia but by host specialisation in the species occurring sympatrically in Europe and Asia. Both specialist and generalist host use strategies have evolved in the group. Although teleomorphs are known in most of the species and unnamed lineages, analyses of the five-gene regions suggest the prevalence of clonal reproduction in H. odoratus. This can be the reason for its success in mushroom farms, also facilitating the spread of fungicide resistance. While tef1 and rpb2 can be recommended for species delimitation, low variation, not exceeding 1 % in the whole ingroup, impeaches the use of ITS as a barcoding gene region in this group of fungi.     

Differential patterns of dehydroabietic acid biotransformation by Nicotiana tabacum and Catharanthus roseus cells

The aim of this study was to use whole cell catalysts as tools for modification of selected resin acids in order to obtain value-added functional derivatives. The enzymatic bioconversion capacities of two plant species were tested towards dehydroabietic acid. Dehydroabietic acid (DHA) is an abundant resin acid in conifers, representing a natural wood protectant. It is also one of the constituents found in by-products of the kraft chemical pulping industry. DHA was fed to tobacco (Nicotiana tabacum) and Madagascar periwinkle (Catharanthus roseus) plant cell and tissue cultures and bioconversion product formation was monitored using NMR analysis. Both plant species took up DHA from culture medium, and various types of typical detoxification processes occurred in both cultures. In addition, diverse responses to DHA treatment were observed, including differences in uptake kinetics, chemical modification of added substrate and changes in overall metabolism of the cells. Interestingly, Catharanthus roseus, a host species for pharmaceutically valuable terpenoid indole alkaloids, exhibited a very different bioconversion pattern for exogenously applied DHA than tobacco, which does not possess a terpenoid indole pathway. In tobacco, DHA is readily glycosylated in the carbonyl group, whereas in periwinkle it is proposed that a cytochrome P450-catalyzed enzymatic detoxification reaction takes place before the formation of glycosylated product.     

Secondary metabolites from Eurotium species, Aspergillus calidoustus and A. insuetus common in Canadian homes with a review of their chemistry and biological activities

As part of studies of metabolites from fungi common in the built environment in Canadian homes, we investigated metabolites from strains of three Eurotium species, namely E. herbariorum, E. amstelodami, and E. rubrum as well as a number of isolates provisionally identified as Aspergillus ustus. The latter have been recently assigned as the new species A. insuetus and A. calidoustus. E. amstelodami produced neoechinulin A and neoechinulin B, epiheveadride, flavoglaucin, auroglaucin, and isotetrahydroauroglaucin as major metabolites. Minor metabolites included echinulin, preechinulin and neoechinulin E. E. rubrum produced all of these metabolites, but epiheveadride was detected as a minor metabolite. E. herbariorum produced cladosporin as a major metabolite, in addition to those found in E. amstelodami. This species also produced questin and neoechinulin E as minor metabolites. This is the first report of epiheveadride occurring as a natural product, and the first nonadride isolated from Eurotium species. Unlike strains from mainly infection-related samples, largely from Europe, neither ophiobolins G and H nor austins were detected in the Canadian strains of A. insuetus and A. calidoustus tested, all of which had been reported from the latter species. TMC-120 A, B, C and a sesquiterpene drimane are reported with certainty for the first time from indoor isolates, as well as two novel related methyl isoquinoline alkaloids.     

Chemistry and pharmacology of Rhaponticum carthamoides: A review

Rhaponticum carthamoides (Willd.) Iljin is a perennial herb, commonly known as a maral root or Russian leuzea, which has been used for centuries in eastern parts of Russia for its marked medicinal properties. This review based on 117 literary sources, with many of them being originally published in non-English languages (mainly in Russian), discusses the current knowledge of traditional uses, chemistry, biological effects and toxicity of this species. Several different classes of compounds were previously isolated from various parts of R. carthamoides of which the main groups are steroids, particularly ecdysteroids, and phenolics (flavonoids and phenolic acids) accompanied with polyacetylenes, sesquiterpene lactones, triterpenoid glycosides and terpenes (essential oil). A comprehensive account of the chemical constituents is given in this review (figures of 120 structures are shown). Various types of preparations, extracts and individual compounds derived from this species have been found to possess a broad spectrum of pharmacological effects on several organs such as the brain, blood, cardiovascular and nervous systems as well as on different biochemical processes and physiological functions including proteosynthesis, work capacity, reproduction, and sexual function. Moreover, the extracts and preparations from the plant, which are hopefully safe, exhibited various additional biological effects e.g. antioxidant, immunomodulatory, anticancerogenic, antimicrobial, antiparasitic and insect antifeedant or repellent activities. The results of data analysis on the chemical, pharmacological and toxicological characteristics of R. carthamoides support the view that this species has beneficial therapeutic properties and indicate its potential as an effective adaptogenic herbal remedy. Finally, some suggestions for further research on chemical and pharmacological properties are given in this review.     

Insecticidal sesquiterpene pyridine alkaloids from Euonymus species

Three insecticidal sesquiterpene pyridine alkaloids with a beta-dihydroagarofuran sesquiterpene skeleton, euoverrine A (1), B (2), and euophelline (3), and a known compound, euojaponine C (4), were isolated from the root bark of Euonymus verrucosides, E. fortunei and E. phellomana by bioassay-guided fractionation. Their chemical structures were elucidated mainly by analyses NMR and MS spectral data.     

Composition and stereochemistry of ephedrine alkaloids accumulation in Ephedra sinica Stapf

Ephedra sinica Stapf (Ephedraceae) is a widely used Chinese medicinal plant (Chinese name: Ma Huang). The main active constituents of E. sinica are the unique and taxonomically restricted adrenergic agonists phenylpropylamino alkaloids, also known as ephedrine alkaloids: (1R,2S)-norephedrine (1S,2S)-norpseudoephedrine, (1R,2S)-ephedrine, (1S,2S)-pseudoephedrine, (1R,2S)-N-methylephedrine and (1S,2S)-N-methylpseudoephedrine. GC–MS analysis of freshly picked young E. sinica stems enabled the detection of 1-phenylpropane-1,2-dione and (S)-cathinone, the first two putative committed biosynthetic precursors to the ephedrine alkaloids. These metabolites are only present in young E. sinica stems and not in mature stems or roots. The related Ephedra foemina and Ephedra foliata also lack ephedrine alkaloids and their metabolic precursors in their aerial parts. A marked diversity in the ephedrine alkaloids content and stereochemical composition in 16 different E. sinica accessions growing under the same environmental conditions was revealed, indicating genetic control of these traits. The accessions can be classified into two groups according to the stereochemistry of the products accumulated: a group that displayed only 1R stereoisomers, and a group that displayed both 1S and 1R stereoisomers. (S)-cathinone reductase activities were detected in E. sinica stems capable of reducing (S)-cathinone to (1R,2S)-norephedrine and (1S,2S)-norpseudoephedrine in the presence of NADH. The proportion of the diastereoisomers formed varied according to the accession tested. A (1R,2S)-norephedrine N-methyltransferase capable of converting (1R,2S)-norephedrine to (1R,2S)-ephedrine in the presence of S-adenosylmethionine (SAM) was also detected in E. sinica stems. Our studies further support the notion that 1-phenylpropane-1,2-dione and (S)-cathinone are biosynthetic precursors of the ephedrine alkaloids in E. sinica stems and that the activity of (S)-cathinone reductases directs and determines the stereochemical branching of the pathway. Further methylations are likely due to N-methyltransferase activities.     

Metabolite profiling of alkaloids and strictosidine synthase activity in camptothecin producing plants

Camptothecin derivatives are clinically used anti-neoplastic alkaloids that biogenetically belong to monoterpenoid indole alkaloids. Camptothecin-related alkaloids from the methanol extracts of Ophiorrhiza pumila, Camptotheca acuminata and Nothapodytes foetida plants were profiled and identified using a reverse-phase high performance liquid chromatography coupled with on-line photodiode array detection and electrospray-ionization ion-trap mass spectrometry. A natural 10-glycosyloxy camptothecin, chaboside, was accumulated in tissues of O. pumila but not in C. acuminata and N. foetida. Anthraquinones regarded as phytoalexins were present in the extracts of hairy roots and calli but not in the differentiated plants of O. pumila. These findings demonstrated a remarkable difference in the constituents between the differentiated plants and the hairy roots or calli tissues. The activity of strictosidine synthase, a key enzyme of camptothecin biosynthesis, was detected in the protein extracts of stems and roots of O. pumila, being correlated with the pattern of strictosidine synthase mRNA expression.     

Dehydrocostus lactone is exuded from sunflower roots and stimulates germination of the root parasite Orobanche cumana

The germination of the obligate root parasites of the Orobanchaceae depends on the perception of chemical stimuli from host roots. Several compounds, collectively termed strigolactones, stimulate the germination of the various Orobanche species, but do not significantly elicit germination of Orobanche cumana, a specific parasite of sunflower.Phosphate starvation markedly decreased the stimulatory activity of sunflower root exudates toward O. cumana, and fluridone - an inhibitor of the carotenoid biosynthesis pathway - did not inhibit the production of the germination stimulant in both shoots and roots of young sunflower plants, indicating that the stimulant is not a strigolactone.We identified the natural germination stimulant from sunflower root exudates by bioassay-driven purification. Its chemical structure was elucidated as the guaianolide sesquiterpene lactone dehydrocostus lactone (DCL). Low DCL concentrations effectively stimulate the germination of O. cumana seeds but not of Phelipanche aegyptiaca (syn. Orobanche aegyptiaca). DCL and other sesquiterpene lactones were found in various plant organs, but were previously not known to be exuded to the rhizosphere where they can interact with other organisms.     

Characterisation of alkaloids from some Australian Stephania (Menispermaceae) species

Chemical investigations of some Stephania species native to Australia and reportedly employed by Aboriginal people as therapeutic agents, are described. The alkaloids from the forest vines Stephania bancroftii F.M. Bailey and S. aculeata F.M. Bailey (Menispermaceae) have been isolated and characterised. The major alkaloids in the tuber of the former species are (-)-tetrahydropalmatine and (-)-stephanine, whereas these are minor components in the leaves, from which a C-7 hydroxylated aporphine has been identified. The major tuber alkaloids in S. aculeata are (+)-laudanidine, and the morphinoid, (-)-amurine, whose absolute stereochemistry has been established by X-ray structural analysis of the methiodide derivative. No significant levels of alkaloids were detected in S. japonica. Complete and unambiguous 1H and 13C NMR data are presented for these alkaloids.