Miniaturized Analytical Strategy Based on µ-SPEed for Monitoring the Occurrence of Pyrrolizidine and Tropane Alkaloids in Honey.

Currently, the analysis of trace-level contaminants in food must be addressed following green analytical chemistry principles and with a commitment to the sustainable development goals. Accordingly, a sustainable and ecofriendly microextraction procedure based on µ-SPEed followed by ultrahigh liquid chromatography coupled to ion-trap tandem mass spectrometry analysis was developed to determine the occurrence of pyrrolizidine and tropane alkaloids in honey samples. The µ-SPEed procedure took approximately 3 min per sample, using only 100 µL of organic solvent and 300 µL of diluted sample. The method was properly validated (overall recoveries 72-100% and precision RSD values ≤15%), and its greenness was scored at 0.61 out of 1. The method was applied to different honey samples, showing overall contamination levels from 32 to 177 µg/kg of these alkaloids. Atropine was found in all the samples, whereas retrorsine N-oxide, lasiocarpine, echimidine, and echimidine N-oxide were the main pyrrolizidine alkaloids in the samples analyzed.

Targeted isolation and identification of bioactive pyrrolidine alkaloids from Codonopsis pilosula using characteristic fragmentation-assisted mass spectral networking.

Codonopsis pilosula (CP), a well-known food medicine homology plant, is commonly used in many countries. In our preliminary study, a series of pyrrolidine alkaloids with high MS responses were detected as characteristic absorbed constituents in rat plasma after oral administration of CP extract. However, their structures were unclear due to the presence of various isomers and the lack of reference standards. In the present study, an MS-guided targeted isolation of pyrrolidine alkaloids of CP extract was performed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF MS). For data analysis under fast data directed acquisition mode (Fast-DDA), an effective approach named characteristic fragmentation-assisted mass spectral networking was successfully applied to discover new pyrrolidine alkaloids with high MS response in CP extract. As a result, seven new pyrrolizidine alkaloids [codonopyrrolidiums C-I (3-9)], together with two known ones (1 and 2), were isolated and identified by NMR spectral analysis. Among them, codonopyrrolidium B (1), codonopyrrolidium D (4) and codonopyrrolidium E (5) were evaluated for lipid-lowering activity, and they could improve high fructose-induced lipid accumulation in HepG2 cells. In addition, the characteristic MS/MS fragmentation patterns of these pyrrolizidine alkaloids were investigated, and 17 pyrrolidine alkaloids were identified. This approach could accelerate novel natural products discovery and characterize a class of natural products with MS/MS fragmentation patterns from similar chemical scaffolds. The research also provides a chemical basis for revealingin vivo effective substances in CP.

Integrative Metabolomics and Proteomics Detected Hepatotoxicity in Mice Associated with Alkaloids from Eupatorium fortunei Turcz.

The traditional Chinese herbal medicine Eupatorium fortunei Turcz. (E. fortunei) has been widely adopted to treat nausea, diabetes, siriasis, and poor appetite. However, E. fortunei contains multiple pyrrolizidine alkaloids (PAs). This study aimed to investigate the hepatotoxicity of total alkaloids in E. fortunei (EFTAs) and identify the toxic mechanisms of EFTAs on hepatocytes. Liquid chromatography with a tandem mass spectrometry assay with reference standards indicated that EFTAs mainly consisted of eight PAs whose content accounted for 92.38% of EFTAs. EFTAs markedly decreased mouse body and liver weights and increased the contents of AST and ALT. The histopathological assays demonstrated that, after exposition to EFTAs, the structures of hepatocytes were damaged and the fibrosis and apoptosis in hepatocytes were accelerated. Moreover, EFTAs increased the serum level of inflammatory cytokines and aggravated circulating oxidative stress. A combination of hepatic proteomics and metabolomics was used to investigate the toxic mechanisms of EFTAs. The study revealed that EFTAs seriously disrupted glycerophospholipid metabolism by upregulating the contents of lysophosphatidylglycerol acyltransferase 1 and phosphatidylinositol and downregulating the contents of choline/ethanolamine kinase beta, choline-ethanolamine phosphotransferase 1, phospholipase D4, 1-acylglycerophosphocholine, phosphatidylcholine, and dihydroxyacetone phosphate in the liver, resulting in detrimental inflammation, fibrosis, and apoptosis. This study revealed that EFTAs induced severe hepatotoxicity by disrupting glycerophospholipid metabolism.

Fate of Food-Relevant Toxic Plant Alkaloids during Food Processing or Storing and Analytical Strategies to Unveil Potential Transformation Products.

Toxic plant alkaloids (TPAs) may contaminate food and pose a threat to consumer health; as a consequence, they are undesired in foodstuff or food commodities. Similar to other ingredients, TPA may be affected by storing or processing of food, often associated with decreased levels of the parent alkaloids. Up to now, little is known about potential transformation products or if they still may exhibit toxic potential to consumers. This perspective briefly summarizes the current knowledge regarding the behavior of opium, pyrrolizidine, and tropane alkaloids toward processing or storing and highlights analytical strategies to identify and elucidate potential transformation products.

Study of the occurrence of toxic alkaloids in forage grass by liquid chromatography tandem mass spectrometry.

The toxic alkaloids in forage grass present a serious health hazard to humans and livestock, especially ergot alkaloids (EAs), pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs). Hence, there is a need for a simultaneous method that allows these dangerous plant toxins to be determined. A simple and effective method was developed to determine fifteen toxic alkaloids (EAs, PAs and TAs) in forage grass using the QuEChERS method and liquid chromatography tandem mass spectrometry (LC-MS/MS). The developed method was validated with average recoveries ranging from 63.10 to 102.10%, and relative standard deviations lower than or equal to 6.39% were obtained. Good linearity over the concentration range of 10-600 µg/kg dry matter (DM) was observed for the target alkaloids. The determination coefficients R2 calculated for each of the matrix calibration curves were greater than 0.99. The limits of detection and quantification were 5 µg/kg DM and 10 µg/kg DM, respectively. The reproducibility of the method was verified in three laboratories: all of the mean recoveries of 15 alkaloids were higher than 60%, and the relative standard deviations in alkaloids between laboratories were all less than 14.24%. The proposed method was applied to analyse 134 forage grass samples from the meadow steppe of Inner Mongolia to monitor toxic alkaloids. A significant difference in the frequency of contamination was found between different herbage species and different regions.

Pyrrolizidine-Derived Alkaloids: Highly Toxic Components in the Seeds of Crotalaria cleomifolia Used in Popular Beverages in Madagascar.

Seeds of Crotalaria cleomifolia (Fabaceae) are consumed in Madagascar in preparation of popular beverages. The investigation of extracts from the seeds of this species revealed the presence of high amounts of alkaloids from which two pyrrolizidine-derived alkaloids were isolated. One of them was fully characterized by spectroscopic and spectrometric methods, which was found to be usaramine. Owing to the high toxicity of these alkaloids, issuing a strong warning among populations consuming the seeds of Crotalaria cleomifolia must be considered.

Development of a new LC-MS method for accurate and sensitive determination of 33 pyrrolizidine and 21 tropane alkaloids in plant-based food matrices.

Setting of maximum limits for a number of plant alkaloids is under discussion in the EU. The novel method developed and optimized in this study enables simultaneous determination of 21 tropane alkaloids (TAs) and 33 pyrrolizidine (PAs) together with their N-oxides (PANOs). For analysis of aqueous-methanolic extract, reversed phase ultra-high-performance liquid chromatography and tandem mass spectrometry (RP-U-HPLC-MS/MS) was employed. The method was validated for frequently contaminated matrices (i) sorghum, (ii) oregano, and (iii) mixed herbal tea. The recoveries at two spiking levels were in the range of 82-115%, 80-106%, and 78-117%, respectively, and repeatabilities were less than 19% for all analyte/matrix combinations. As regards the achieved limits of quantification (LOQ), their values were in the range of 0.5-10 µg kg-1. The crucial problem encountered during method development, co-elution of multiple groups of isomeric alkaloids, was overcome by subsequent sample separation in the second chromatographic system, hydrophilic interaction liquid chromatography (HILIC), providing different separation selectivity. Lycopsamine, echinatine, and indicine (co-elution group 1) and N-oxides of indicine and intermedine (co-elution group 2), which could not be resolved on the commonly used RP column, were possible to separate fully by using the HILIC system.

Genetic diversity, reproductive capacity and alkaloids content in three endemic Alkanna species.

The Balkans endemic species Alkanna primuliflora Griseb., A. stribrnyi Velen., and A. graeca Boiss. & Spruner have limited distribution in the Balkan Peninsula and a large variation in the morphological characteristics. The populations of the three Alkanna species in the Bulgarian flora are small and fragmented. There are no previous reports on the chemical profile or on the embryology of these species. The hypothesis was that the limited distribution of A. primuliflora, A. stribrnyi, and A. graeca was due to their reproductive capacity and genetic diversity. Furthermore, we hypothesized that the three species will contain pyrrolizidine alkaloids (PAs), as other species of the genus Alkanna (Boraginaceae), but they would have differential alkaloids composition. The population genetic structure and differentiation showed a clear distinction between species and revealed average levels of genetic diversity among the natural populations of the three Alkanna species. The embryological investigation observed stability of the processes in the male and female generative spheres and high viability of mature pollen and embryo of the three species. The normal formation of male and female gametophytes without deviations or degenerative processes, and observed levels of genetic diversity between Alkanna individuals are important in maintaining the size and resilience of the Alkanna populations. Eight alkaloids were identified by GC-MS in A. primuliflora and A. graeca and six alkaloids in A. stribrnyi. The main pyrrolizidine alkaloids (PAs) in all investigated species was triangularine. A. primuliflora and A. graeca showed similar chemical composition that comprised 9-angeloylretronecine, 7-tigloylretronecine, 9-tigloylretronecine, triangularicine, dihydroxytriangularine, dihydroxytriangularicine, whereas, in A. stribrnyi 9-tigloylretronecine, triangularicine and dihydroxytriangularicine were not found. This is the first report on the presence of PAs in A. primuliflora, A. stribrnyi and A. graeca. Besides, this is the first report on the embryology of these endemic species. The results contribute to the knowledge of the three endemic Alkanna species and will facilitate policy-making and defining new strategies for their conservation.

Alkaloid profiling of herbal drugs using high resolution mass spectrometry.

Herbal infusions are consumed worldwide thanks to their "natural" beneficial effects, also due to the presence of alkaloids, although these compounds can have poisonous effects. A method combining online solid-phase purification with high resolution mass spectrometry was used to define the alkaloid profiles of 117 herbs and 7 commercial blends. Forty-one alkaloids were quantified in reference to analytical standards, while the presence of a further 116 was confirmed based on accurate mass, retention time, and fragmentation profile. The targeted study showed that 52% of herbs and 42% of commercial blends contained at least one alkaloid. Pyrrolizidines were the most commonly present (26% of samples), with concentrations generally ranging from the quantification limit to roughly 100 µg kg-1 . Moreover, a homemade infusion was studied, finding on average 45% and 6% lower extraction for pyrrolizidine and steroidal alkaloids, respectively. Nevertheless, the migration of pyrrolizidines was confirmed. The study confirmed the frequent presence, natural or accidental, of alkaloids in commercial infusion herbs, highlighting the urgent need for routine and accurate controls.

Alkaloids in the human food chain--natural occurrence and possible adverse effects.

Alkaloid-containing plants are an intrinsic part of the regular Western diet. The present paper summarizes the occurrence of alkaloids in the food chain, their mode of action and possible adverse effects including a safety assessment. Pyrrolizidine alkaloids are a reason for concern because of their bioactivation to reactive alkylating intermediates. Several quinolizidine alkaloids, ß-carboline alkaloids, ergot alkaloids and steroid alkaloids are active without bioactivation and mostly act as neurotoxins. Regulatory agencies are aware of the risks and have taken or are considering appropriate regulatory actions for most alkaloids. These vary from setting limits for the presence of a compound in feed, foods and beverages, trying to define safe upper limits, advising on a strategy aiming at restrictions in use, informing the public to be cautious or taking specific plant varieties from the market. For some alkaloids known to be present in the modern food chain, e.g., piperine, nicotine, theobromine, theophylline and tropane alkaloids risks coming from the human food chain are considered to be low if not negligible. Remarkably, for many alkaloids that are known constituents of the modern food chain and of possible concern, tolerable daily intake values have so far not been defined.

Degradation of yew, ragwort and rhododendron toxins during composting.

Recent concerns have been raised that plants such as ragwort (Senecio jacobaea), yew (Taxus baccata) and rhododendron (Rhododendron ponticum) that are toxic to livestock may be included in compost windrows but may not be fully detoxified by the composting process. This study investigates the decomposition during composting of toxic pyrrolizidine alkaloids present in ragwort, taxines (A and B) present in yew, and grayanotoxins (GTX I, II, and III) present in rhododendron during composting. Plant samples were contained within microporous bags either towards the edge or within the centre of a pilot-scale compost heap. They were destructively harvested at regular intervals over 1200 degrees C cumulative temperature (about three months). Samples were analysed for levels of toxins by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). Pyrrolizidine alkaloids and taxines were shown to degrade completely during the composting process. While GTX I showed significant reductions, concentrations of GTX III remained unchanged after 1200 degrees C cumulative temperature. However, estimates of exposure to grazing livestock coming into contact with source-segregated green waste compost containing up to 7% rhododendron suggest that GTX III poses no appreciable risk.

Determination of senkirkine and senecionine in Tussilago farfara using microwave-assisted extraction and pressurized hot water extraction with liquid chromatography tandem mass spectrometry.

Tussilago farfara (Kuan Donghua) is an important Chinese herbal medicine which has been shown to contain many bioactive compounds and widely used to relieve cough and resolve phlegm. However, besides therapeutic bioactive compounds, this herb has been found to contain toxic pyrrolizidine alkaloids (PAs), mainly senkirkine and traces of senecionine. In this report, conditions for microwave-assisted extraction (MAE) and pressurized hot water extraction (PHWE) were optimized for the extraction of the PAs. The results were compared against heating under reflux. It was found that the binary mixture of MeOH:H(2)O (1:1) acidified using HCl to pH 2-3 was the optimal solvent for the extraction of the PAs in the plant materials. Liquid chromatography (LC) with ultra-violet (UV) detection and electrospray ionization mass spectrometry (ESI-MS) in the positive mode was used for the determination and quantitation of senkirkine and senecionine in the botanical extract. The proposed extraction methods with LC/MS allow for the rapid detection of the major and the minor alkaloids in T. farfara in the presence of co-eluting peaks. With LC/MS, the quantitative analysis of PAs in the extract was done using internal standard calibration and the precision was found to vary from 0.6% to 5.4% on different days. The limits of detection (LODs) and limits of quantitation (LOQs) for MAE and PHWE were found to vary from 0.26 microg/g to 1.04 micro/g and 1.32 micro/g to 5.29 microg/g, respectively. The method precision of MAE and PHWE were found to vary from 3.7% to 10.4% on different days. The results showed that major and minor alkaloids extracted using MAE and PHWE were comparable to that by heating under reflux. Our data also showed that significant ion suppression was not observed in the analysis of senkirkine and senecionine in the botanical extracts with co-eluting peaks.

Insect-synthesised retronecine ester alkaloids: precursors of the common arctiine (Lepidoptera) pheromone hydroxydanaidal.

Many pyrrolizidine alkaloid (PA)-adapted insects convert PAs sequestered from their larval host plants into "insect-PAs" in which the acid components of the alkaloids are replaced by small, branched aliphatic 2-hydroxy acids of insect origin. It has been proposed that insect-PAs are precursors of the pheromone hydroxydanaidal in male Estigmene acrea moths, but it is not clear why they are specifically required or what the structural features or chemical properties are that make insect-PAs more suitable for conversion into hydroxydanaidal than superficially similar alkaloids of plant origin. Evidence is presented that insect-PAs are also precursors of hydroxydanaidal in the polyphageous arctiine, Creatonotos transiens, and a new biosynthetic pathway to hydroxydanaidal is proposed that has a mandatory requirement for insect-PAs as intermediates.

On the sequence of bond formation in loline alkaloid biosynthesis.

Loline alkaloids are saturated pyrrolizidines with an oxygen bridge between carbon atoms C-2 and C-7 and an amino group on C-1. They are bioprotective alkaloids produced by Epichloë and Neotyphodium species, mutualistic fungal endophytes that are symbiotic with cool-season grasses. The sequence of bond formation in loline alkaloid biosynthesis was determined by synthesizing deuterated forms of potential intermediates and feeding them to cultures of the endophyte Neotyphodium uncinatum. These cultures incorporated deuterium from labeled N-(3-amino-3-carboxypropyl)proline and exo-1-aminopyrrolizidine into N-formylloline. The first result suggests that N-(3-amino-3-carboxypropyl)proline is the first committed intermediate in loline biosynthesis, and the second result demonstrates that the pyrrolizidine rings form before the ether bridge. The incorporation of these two compounds into lolines and the lack of incorporation of several related compounds clarify the order of bond formation in loline alkaloid biosynthesis.

Investigation of pyrrolizidine alkaloids and their N-oxides in commercial comfrey-containing products and botanical materials by liquid chromatography electrospray ionization mass spectrometry.

Pyrrolizidine alkaloids (PAs) and their N-oxides are found in several plant families throughout the world. PAs are potentially toxic to the liver and/or lungs in humans and may cause acute liver failure, cirrhosis, pneumonitis, or pulmonary hypertension. PAs are also carcinogenic to animals, and they have been linked to the development of hepatocellular and skin squamous cell carcinomas as well as liver angiosarcomas. According to experimental studies, the quantity of PAs in some herbal teas and dietary supplements is sufficient to be carcinogenic in exposed individuals. A method for the extraction and identification of PAs and their N-oxides in botanical materials and commercial comfrey-containing products has been developed using liquid chromatography electrospray ionization mass spectrometry. Following optimization of the extraction procedure and the chromatographic conditions, the method was applied to the analysis of 10 herbal remedies. All of the products that were labeled to contain comfrey were found to contain measurable quantities of PAs.

Polyhydroxylated pyrrolidine and pyrrolizidine alkaloids from Hyancinthoides non-scripta and Scilla campanulata.

Aqueous ethanol extracts from the immature fruits and stalks of bluebell (Hyacinthoides non-scripta) were subjected to various ion-exchange column chromatographic steps to give 1,4-dideoxy-1,4-imino-D-arabinitol (1),2(R),5(R)-bis(hydroxymethyl)-3(R),4(R)-dihydroxypyrrolidine (DMDP) (2), 6-deoxy-6-C-(2,5-dihydroxyhexyl)-DMDP (3),2,5-dideoxy-2,5-imino-DL-glycero-D-manno-heptitol (homoDMDP)(4),homoDMDP-7-O-apioside (5), homoDMDP-7-O-beta-D-xylopyranoside (6), (1S*,2R*,3R*,5R*,7aR*)-1,2-dihydroxy-3,5- dihydroxymethylpyrrolizidine (7), and (1S*,2R*,3R*,5R*,6R*,7R*,7aR*)-3-hydroxymethyl-5-methyl-1,2,6,7 tetrahydroxypyrrolizidine (8). Bulbs of Scilla campanulata (Hyacinthaceae) yielded (1S*,2R*,3R*,5S*,7aR*)-1,2-dihydroxy-3,5-dihydroxy-methylpyrrol izidine (9) in addition to compounds 1-7. Compounds 3,6,7,8, and 9 are new natural products. Compound 4 is a potent competitive inhibitor with K(i) values of 1.5 microM for Caldocellum saccharolyticum beta-glucosidase and 2.2 microM for bovine liver beta-galactosidase. The 7-O-beta-D xyloside 6 was a stronger competitive inhibitor than 4 of C saccharolyticum beta-glucosidase and rat intestinal lactase, with K(i) values of 0.06 and 0.07 microM, respectively, but a weaker inhibitor of bovine liver beta-galactosidase. Furthermore, compound 4 is also a competitive inhibitor (K(i) = 1.8 microM) of porcine kidney trehalase, but 6 was inactive against this enzyme.

The comparative metabolism of the four pyrrolizidine alkaloids, seneciphylline, retrorsine, monocrotaline, and trichodesmine in the isolated, perfused rat liver.

Despite their similarity in structure, pyrrolizidine alkaloids (PAs) vary in their LD50s and in the organs in which toxicity is expressed. We have examined whether there are differences in the metabolism of certain PAs that are associated with these quantitative and qualitative differences in toxicity. Isolated rat livers were perfused with one of four PAs (seneciphylline, retrorsine, monocrotaline, and trichodesmine) at 0.5 mM for 1 hr, and the pyrrolic metabolites determined that were released into perfusate and bile or bound in the liver. The proportion of the PA removed by the liver varied from 93% for retrorsine to 55% for trichodesmine. However, trichodesmine-perfused livers released the greatest amount of the dehydroalkaloid into the perfusate. These reactive pyrrolic metabolites appear to be largely responsible for the toxicity of PAs. Over the course of a 1-hr perfusion, dehydroalkaloid release varied fourfold among the PAs examined. Seneciphylline and retrorsine significantly increased bile flow. Highest concentrations of PAs in bile were achieved at 30-40 min perfusion. Conversion of dehydroalkaloid to the conjugate 7-glutathionyl-6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine (GSDHP) is a detoxification reaction. GSDHP release into bile varied from 80 nmol/g liver for trichodesmine to 880 nmol/g for retrorsine. Release of the less toxic hydrolytic product of dehydroalkaloids, 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, was also determined. Bound pyrroles in liver are probably an indication of heptatoxicity. At the end of perfusion these varied from 55 nmol/g for monocrotaline to 195 nmol/g for retrorsine. The chemical form of the bound pyrroles is a 7-thioether conjugate of 6,7-dihydro-1-hydroxymethyl-5H-pyrrolizine. No 7,9-dithio conjugate was detected, indicating that only monoalkylation has been found. These differences in metabolic pattern reflect differences in reactivity of the initially formed dehydroalkaloid and can account for the toxicological differences between the parent PAs.

Alkaloids in Madagascan frogs (Mantella): pumiliotoxins, indolizidines, quinolizidines, and pyrrolizidines.

Brightly colored ranid frogs of the genus Mantella are found only in rain forests of Madagascar. Gc-ms and gc-Ft-ir analyses of skin alkaloids of seven different species, including four populations of Mantella madagascariensis, are reported. All contain one or more representatives of the pumiliotoxin A (PTX-A) class with the 13,14-dihydro derivatives 309A and 325A found in major amounts in the four populations of M. madagascariensis, while 307A (PTX-A) is found in two populations of M. madagascariensis and in three additional species, Mantella aurantiaca, Mantella viridis, and Mantella crocea. The latter three species also contain appreciable quantities of 323A (PTX-B). The four populations of M. madagascariensis show major amounts of two 1,4-disubstituted quinolizidines, 217A and 231A, and a 5,8-disubstituted indolizidine, 217B, in addition to many minor or trace quinolizidines and indolizidines. Such disubstituted quinolizidines and indolizidines are present as trace alkaloids in the six other species of Mantella, along with 3,5-disubstituted indolizidines, 3,5-disubstituted pyrrolizidines, the decahydroquinoline cis-195A, tricyclic alkaloids, and homopumiliotoxins. A new alkaloid class, which appears to contain a quinolizidine moiety, is seen in M. aurantiaca and M. crocea and is represented by 235C and several congeners.

Determination of Senecio alkaloids by thermospray liquid chromatography/mass spectrometry.

A series of Senecio alkaloid and alkaloid N-oxide standards has been analyzed using positive and negative ion thermospray liquid chromatography/mass spectrometry (LC/MS) with an ammonium acetate-containing mobile phase. On-line separations of pyrrolizidine alkaloids from extracts of Senecio jacobaea (tansy ragwort) and Senecio vulgaris (common groundsel) were done using an ammonium hydroxide-containing mobile phase. All of the alkaloids known to be present in the extracts were detected by ammonium hydroxide thermospray LC/MS, as well as many other components which may be as-yet-unidentified alkaloids.

Australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing.

Australine [(1R,2R,3R,7S,7aR)-3-(hydroxymethyl)-1,2,7-trihydroxypyrrolizid ine] is a polyhydroxylated pyrrolizidine alkaloid that was isolated from the seeds of the Australian tree Castanospermum australe and characterized by NMR and X-ray diffraction analysis [Molyneux et al. (1988) J. Nat. Prod. (in press)]. Since swainsonine and catanospermine are polyhydroxylated indolizidine alkaloids that inhibit specific glycosidases, we tested australine against a variety of exoglycosidases to determine whether it would inhibit any of these enzymes. This alkaloid proved to be a good inhibitor of the alpha-glucosidase amyloglucosidase (50% inhibition at 5.8 microM), but it did not inhibit beta-glucosidase, alpha- or beta-mannosidase, or alpha- or beta-galactosidase. The inhibition of amyloglucosidase was of a competitive nature. Australine also inhibited the glycoprotein processing enzyme glucosidase I, but had only slight activity toward glucosidase II. When incubated with cultured cells, this alkaloid inhibited glycoprotein processing at the glucosidase I step and caused the accumulation of glycoproteins with Glc3Man7-9(GlcNAc)2-oligosaccharides.