The role of chemical fingerprinting: application to Ephedra

Ephedra sinica, known as Ma Huang, is one of the oldest medicinal herbs in Traditional Chinese Medicine (TCM). Preparations, namely teas, of E. sinica have been used for over 5000 years as a stimulant and as an antiasthmatic. In the West, extracts of E. sinica, E. intermedia or E. equisetina are most commonly used in dietary supplements as a stimulant and to promote weight loss. More than 50 species of Ephedra are native to both hemispheres, but the detection of ephedrine alkaloids has been limited to species in Eurasia. Currently, methods exist to quantitate the ephedrine alkaloids in extracts of plant material or dietary supplements, but the methods are not able to verify the extract is of an Ephedra species. Reverse phase high performance liquid chromatography with photodiode array detection was applied for the chemical fingerprinting of the Ephedra species. Two regions of comparison were determined in the chromatograms at 320 nm. The series of peaks between 52 and 64 min confirms an Ephedra species is being analyzed. The aforementioned peaks also could distinguish between Ephedra species from Eurasia, North America and South America. Peaks at ca. 57 and 59 min were isolated and determined to be two new compounds, 4-(2-eicosyloxycarbonyl-vinyl)-benzoic acid and 4-(2-docosyloxycarbonyl-vinyl)-benzoic acid respectively. Authentication of ground plant material as Ephedra can be achieved by this chemical fingerprinting method.     

Biosynthesis of amphetamine analogs in plants

Amphetamine analogs are produced by plants in the genus Ephedra and by Catha edulis, and include the widely used decongestants and appetite suppressants pseudoephedrine and ephedrine. A combination of yeast (Candida utilis or Saccharomyces cerevisiae) fermentation and subsequent chemical modification is used for the commercial production of these compounds. The availability of certain plant biosynthetic genes would facilitate the engineering of yeast strains capable of de novo pseudoephedrine and ephedrine biosynthesis. Chemical synthesis has yielded amphetamine analogs with myriad functional group substitutions and diverse pharmacological properties. The isolation of enzymes with the serendipitous capacity to accept novel substrates could allow the production of substituted amphetamines in synthetic biosystems. Here, we review the biology, biochemistry and biotechnological potential of amphetamine analogs in plants.     

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。     

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 ephedrines as their carbon disulfide derivatives

A method for the separation and identification of the diastereoisomers ephedrine and pseudoephedrine and the diastereoisomers norephedrine and norpseudoephedrine is presented. The compounds were derivatised by reaction with carbon disulfide in the presence of alkali. These derivatives and their trifluoroacetic anhydride derivatives were subjected to gas chromatography with nitrogen-selective detection as well as mass-selective detection and high-performance liquid chromatography with ultraviolet detection. The results showed that ephedrine and pseudoephedrine can easily be differentiated by gas chromatographic analyses of their carbon disulfide derivatives. Norephedrine and norpseudoephedrine can be differentiated by the different chromatographic retention times of their carbon disulfide derivatives and by the fact that norephedrine yielded two products and norpseudoephedrine only one product when reacted with carbon disulfide under the same conditions. Trifluoroacetylation of the latter compounds gave a more pronounced differentiation.     

A metabolite fingerprinting for the characterization of commercial botanical dietary supplements

Phytopharmaceuticals, phytomedicines and botanical dietary supplements are products of wide interest considering the increase of their use. The development of fast and effective analytical methods able to give a fingerprinting of the product, on the basis of the plant extracts declared to be contained in it, is surely of high interest. In a previous investigation electrospray mass spectrometry was proved to be effective for the characterization of plant extracts. The direct infusion of the samples and the analyses in both positive and negative ion mode lead to a clear differentiation of the different samples. To verify if the same approach can be effective also for mixtures of plant extracts, five different commercial dietary supplements [Sedivitax gocce (1), Finocarbo Plus opercoli (2), Sollievo Bio tavolette (3), MiniMas opercoli (4) and Ruscoven gocce (5), all products from Aboca S.p.A., Sansepolcro, Italy] were analyzed by ESI. In order to evaluate possible changes in the metabolic profile with respect to different years of production, ten different batches of the commercial dietary supplements were considered. The mass spectral data were evaluated by multivariate analysis and the obtained results suggest that the method allows a satisfactory and rapid characterization of complex mixtures of commercial dietary supplements.     

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.     

盐碱地种植麻黄试验研究

麻黄在干旱区硫酸盐,氯化物盐碱地中种植,其土壤0-30cm土层含盐量1.2%以下生长良好,初步化验分析,麻黄在盐碱地中种植其麻黄碱含量有所提高,同时可获得较大的社会、生态、经济效益。     

Risk Associated with the Use of Selected Ingredients in Food Supplements

This review focuses on four new product categories of food supplements: pre-workout, fat burner/thermogenic, brain/cognitive booster, and hormone/testosterone booster. Many food supplements have been shown to be contaminated with unauthorized substances. In some cases, the ingredients in the new categories of dietary supplements were medicinal products or new synthetic compounds added without performing clinical trials. Some of the new ingredients in dietary supplements are plant materials that are registered in the pharmacopoeia as herbal medicines. In other cases, dietary supplements may contain plant materials that have no history of human use and are often used as materials to ‘camouflage’ stimulants. In the European Union, new ingredients of dietary supplements, according to European Food Safety Authority or unauthorized novel food. Furthermore, selected ingredients in dietary supplements may be prohibited in sports and are recognized as doping agents by World Anti-Doping Agency.     

Separation of ephedrine and pseudoephedrine enantiomers using a polysaccharide‐based chiral column: A normal phase liquid chromatography–high‐resolution mass spectrometry approach

Chiral considerations are found to be very much relevant in various aspects of forensic toxicology and pharmacology. In forensics, it has become increasingly important to identify the chirality of doping agents to avoid legal arguments and challenges to the analytical findings. The scope of this study was to develop an liquid chromatography–mass spectrometry (LCMS) method for the enantiomeric separation of typical illicit drugs such as ephedrines (ie, 1S,2R(+)‐ephedrine and 1R,2S(?)‐ephedrine) and pseudoephedrine (ie, R,R(?)‐pseudoephedrine and S,S(+)‐pseudoephedrine) by using normal phase chiral liquid chromatography–high‐resolution mass spectrometry technique. Results show that the Lux i‐amylose‐1 stationary phase has very broad and balancing‐enantio‐recognition properties towards ephedrine analogues, and this immobilized chiral stationary phase may offer a powerful tool for enantio‐separation of different types of pharmaceuticals in the normal phase mode. The type of mobile phase and organic modifier used appear to have dramatic influences on separation quality. Since the developed method was able to detect and separate the enantiomers at very low levels (in pico grams), this method opens easy access for the unambiguous identification of these illicit drugs and can be used for the routine screening of the biological samples in the antidoping laboratories.     

Ligands of cholinesterases of ephedrine and pseudoephedrine structure

The paper is a review of literature data on interaction of erythrocytic acetylcholinesterase and of mammalian blood serum butyrylcholinesterase with a group of isomeric complex ester derivatives (acetates, propionates, butyrates, valerates, and isobutyrates) of bases and iodomethylates of ephedrine and its enantiomer pseudoephedrine. For 20 alkaloid monoesters, parameters of enzymatic hydrolysis are determined and their certain specificity toward acetylcholinesterase is revealed, whereas 5 diesters of iodomethylates of pseudoephedrine were submitted to hydrolysis only by butyrylcholinesterase. It turned out that 20 alkaloid diesters and 10 trimethylammonium derivatives were uncompetitive reversible inhibitors of acetylcholinesterase and competitive inhibitors of butyrylcholinesterase. The performed for the first time isomer and enantiomer analysis “structure—efficiency” has shown that in most caes it is possible to state the greater complementarity of catalytical surface of enzymes for ligands of pseudoephedrine structure, such differentiation being more often manifested.     

Botanicals authentication in food,food supplements and herbal medicinal products

Nowadays, there is a constant increase of health products (food supplements, medical devices and so on), in the market, despite the crisis that involves a lot of nations at the international level. The plants and their derivatives are present in high percentage in these products and moreover people usually have great trust in plants and in natural products in general. The above-mentioned products are available in the market in the form of tablets, pills, lozenges, syrups and other forms that supplement our diet (food or dietary supplements) or act with a therapeutic effect (herbal medicines). In Italy, about 50% of the sold products contain plants and/or plant extracts, better known as botanicals according to the European Food Safety Authority settlement. It is therefore understandable how the botanicals adulteration and/or the sophistication can heavily weigh on the quality of the finished products, not only from an economic point of view but also in guaranteeing the safety of consumers.     

Solid-phase extraction and HPLC-MS profiling of pyrrolizidine alkaloids and their N-oxides: a case study of Echium plantagineum

Pyrrolizidine alkaloids and their N-oxides can be extracted from the dried methanolic extracts of plant material using dilute aqueous acid. The subsequent integration of solid-phase extraction (with a strong cation exchanger) of the alkaloids and N-oxides from the aqueous acid solution, together with analysis using HPLC-ESI/MS, provides a method for the simultaneous profiling of the pyrrolizidine alkaloids and their N-oxides in plant samples and the collection of useful structural data as an aid in their identification. The N-oxide character of the analytes may be confirmed by treating analytical samples with a redox resin and observing the formation of the corresponding parent pyrrolizidine alkaloids. The present case study of Echium plantagineum highlighted a higher ratio of N-oxides to the parent tertiary bases than has been previously reported. Furthermore, a higher proportion of acetylated pyrrolizidine-N-oxides was observed in the flower heads relative to the leaves. Six pyrrolizidine alkaloids or pyrrolizidine-N-oxides, not previously reported from E. plantagineum, were tentatively identified on the basis of MS and biogenetic considerations. Three of these, 3'-O-acetylintermedine/lycopsamine, leptanthine-N-oxide and 9-O-angelylretronecine-N-oxide, have been reported elsewhere, whilst three others, 3'-O-acetylechiumine-N-oxide, echimiplatine-N-oxide and echiuplatine-N-oxide, appear unreported from any other source.     

Gluten screening of several dietary supplements by immunochromatographic assay

Celiac disease (CD) is a chronic intestinal disorder of public health concern caused by gluten ingestion in sensitive individuals. Gluten is a protein found not only in gluten-containing food but also as normal component of drugs and dietary supplements. Detection of gluten in dietary supplements is a very important task required for establishing their gluten status, which is highly important for the safety of products consumed by CD and gluten-sensitive patients. In this paper, we investigated the presence of gluten in twenty one common dietary supplements from the national market using the immunochromatographic assay. This visual assay proved to be an efficient rapid tool for gluten screening as an alternative to the ELISA techniques. The results have shown the presence of gluten in 23.8% of the investigated samples (vitamins, minerals, plant extracts, probiotics supplements, lactoferrin, propolis supplements). The results provide information which may contribute to the completion of the existing lists of gluten-free pharmaceuticals. It is known that for CD patients obtaining accurate information about the gluten content of a particular item is a difficult and time-consuming process.     

Application of force field calculations to the prediction of chirally discriminating chromatographic behaviour for β-CDs

β-Cyclodextrin and its derivatives have been utilised to effect chiral separation in HPLC and CZE, both as stationary phases and mobile phase additives. The basis of the method is assumed to depend upon the formation of inclusion complexes of differing stabilities between enantiomeric analytes and the cyclodextrin, resulting in a differential dynamic distribution between chromatographic phases. In this study, force field calculations have been employed to model the inclusion complexes of enantiomeric brompheniramine, ephedrine, pseudoephedrine, ibuprofen, mandelic acid, methylphenobarbitone, and hexobarbitone with β-cyclodextrin. The resulting values for Δ(ΔH), the difference in enthalpy of complex formation between enantiomeric pairs has been compared with literature chromatographic data to explain the ability of the systems to achieve enantiomeric separations. © 1996 Wiley-Liss, Inc.     

Simultaneous chiral separation of methylamphetamine and common precursors using gas chromatography/mass spectrometry

Methylamphetamine, ephedrine, and pseudoephedrine were derivatized using trifluoroacetic anhydride and enantiomers of each were analyzed using gas chromatography coupled to mass spectrometry (GC/MS) fitted with a γ‐cyclodextrin (Chiraldex™ G‐PN) chiral column. A temperature‐programmed method was developed and optimized and the results compared with those obtained using a previously published isothermal GC method applied to GC/MS analysis. Trifluoroacetylated 3‐(trifluoromethyl)phenethylamine hydrochloride was used as an internal standard, and mass fragmentation patterns are proposed for all derivatives analyzed. Qualitative validation of the optimized chromatographic conditions was completed in accordance with the guidelines published by the United Nations Office on Drugs and Crime (UNODC). Under conditions of repeatability and reproducibility, the method gave relative retention times with a relative standard deviation of less than 0.02% for all six analytes of interest. This surpasses the UNODC's acceptance criteria of 2% for validation of qualitative precision. Ephedrine and pseudoephedrine are common precursors in the clandestine manufacture of methylamphetamine. Seizures of illicit methylamphetamine therefore often contain mixtures of these optically active compounds. The simultaneous enantioseparation of these compounds to produce a profile would provide valuable information to law enforcement agencies regarding the provenance of a methylamphetamine seizure. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Stability studies of amphetamine and ephedrine derivatives in urine

Knowledge of the stability of drugs in biological specimens is a critical consideration for the interpretation of analytical results. Identification of proper storage conditions has been a matter of concern for most toxicology laboratories (both clinical and forensic), and the stability of drugs of abuse has been extensively studied. This concern should be extended to other areas of analytical chemistry like antidoping control. In this work, the stability of ephedrine derivatives (ephedrine, norephedrine, methylephedrine, pseudoephedrine, and norpseudoephedrine), and amphetamine derivatives (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA)) in urine has been studied. Spiked urine samples were prepared for stability testing. Urine samples were quantified by GC/NPD or GC/MS. The homogeneity of each batch of sample was verified before starting the stability study. The stability of analytes was evaluated in sterilized and non-sterilized urine samples at different storage conditions. For long-term stability testing, analyte concentration in urine stored at 4 degrees C and -20 degrees C was determined at different time intervals for 24 months for sterile urine samples, and for 6 months for non-sterile samples. For short-term stability testing, analyte concentration was evaluated in liquid urine stored at 37 degrees C for 7 days. The effect of repeated freezing (at -20 degrees C) and thawing (at room temperature) was also studied in sterile urine for up to three cycles. No significant loss of the analytes under study was observed at any of the investigated conditions. These results show the feasibility of preparing reference materials containing ephedrine and amphetamine derivatives to be used for quality control purposes.     

Analytical characterisation of crude extracts from an African Ancistrocladus species using high-performance liquid chromatography and capillary electrophoresis coupled to ion trap mass spectrometry

The analysis by HPLC, CE and CE-MS/MS of root bark extracts of a, so far undescribed, Central-African Ancistrocladus species (family Ancistrocladaceae) is described. Owing to the complexity of the extract, the application of reversed-phase HPLC resulted in a partially incomplete separation of the naphthylisoquinoline alkaloids, whilst CE using a non-aqueous buffer proved to be a very valuable complementary method for a first characterisation of the crude extract. By performing additional CE-MS/MS experiments, in combination with parallel isolation studies and structural elucidation using conventional methods, six alkaloidal substances present in the plant could be identified.     

Synthesis of metallic nanoparticles using plant extracts

Biomolecules present in plant extracts can be used to reduce metal ions to nanoparticles in a single-step green synthesis process. This biogenic reduction of metal ion to base metal is quite rapid, readily conducted at room temperature and pressure, and easily scaled up. Synthesis mediated by plant extracts is environmentally benign. The reducing agents involved include the various water soluble plant metabolites (e.g. alkaloids, phenolic compounds, terpenoids) and co-enzymes. Silver (Ag) and gold (Au) nanoparticles have been the particular focus of plant-based syntheses. Extracts of a diverse range of plant species have been successfully used in making nanoparticles. In addition to plant extracts, live plants can be used for the synthesis. Here we review the methods of making nanoparticles using plant extracts. Methods of particle characterization are reviewed and potential applications of the particles in medicine are discussed.     

Aneugenic effects of benzoxazinones in cultured human cells

Benzoxazinones (BAs) are natural products that are present in Gramineae and represent part of the plant defence system against pests. In recent years, sprouts of maize, wheat and rye have been used for the production of dietary supplements. We have investigated the potential genotoxic activities of a diverse range of synthetic derivatives of the most abundant natural BA, namely DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one), proposed for use as a potential herbicide. We have tested 18 synthetic BAs for potential effects in cultured HeLa cells. We found significantly higher micronucleus (MN) induction over the background level, with the solvent DMSO used as an internal control. Concentration-dependent effects were found between 1 nM and 20 nM for all the synthetic compounds studied. Immunostaining with an anticentromere antibody showed that >80% of MN induced gave a centromere-positive signal. Similarly, fluorescence in situ hybridization (FISH) analysis with alphoid centromere probes showed a positive hybridization signal, indicating that all compounds analyzed are aneugenic. Chemical modification of the N in the heterocyclic aromatic amine served us to suggest a relationship between the structure and the aneugenic effects of the compounds analyzed. Our findings indicate that benzoxazinoids could be potential genotoxins for human cells.