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Phytochemical Analysis - Current Research Articles



Current research articles: Phytochemistry

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Phytochemical Analysis - published by Wiley

Phytochemical Analysis devoted to the publication of original articles concerning the development, improvement, validation and/or extension of application of analytical methodology in the plant sciences. The spectrum of coverage is broad, encompassing methods and techniques relevant to the detection (including bio-screening), extraction, separation, purification, identification and quantification of compounds in plant biochemistry, plant cellular and molecular biology, plant biotechnology, the food sciences, agriculture and horticulture




Current articles of the journal:



Purification of Active Myrosinase from Plants by Aqueous Two-Phase Counter-Current Chromatography

Introduction Myrosinase (thioglucoside glucohydrolase; E.C. 3.2.1.147), is a plant enzyme of increasing interest and importance to the biomedical community. Myrosinase catalyses the formation of isothiocyanates such as sulforaphane (from broccoli) and 4-(?-l-rhamnopyranosyloxy)benzyl isothiocyanate (from moringa), which are potent inducers of the cytoprotective phase-2 response in humans, by hydrolysis of their abundant glucosinolate (?-thioglucoside N-hydroxysulphate) precursors. Objective To develop an aqueous two-phase counter-current chromatography (CCC) system for the rapid, three-step purification of catalytically active myrosinase. Methods A high-concentration potassium phosphate and polyethylene glycol biphasic aqueous two-phase system (ATPS) is used with a newly developed CCC configuration that utilises spiral-wound, flat-twisted tubing (with an ovoid cross-section). Results Making the initial crude plant extract directly in the ATPS and injecting only the lower phase permitted highly selective partitioning of the myrosinase complex before a short chromatography on a spiral disk CCC. Optimum phase retention and separation of myrosinase from other plant proteins afforded a 60-fold purification. Conclusion Catalytically active myrosinase is purified from 3-day broccoli sprouts, 7-day daikon sprouts, mustard seeds and the leaves of field-grown moringa trees, in a CCC system that is predictably scalable. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 7 August 2014 | 11:27 am


Advances in Isoflavone Profile Characterisation using Matrix Solid-phase Dispersion Coupled to HPLC/DAD in Medicago Species

Introduction Analytical methods used in phytochemistry analysis are limited by the sample preparation step, which should ideally be fast, accurate, ecofriendly and achievable using low quantities of the sample. Matrix solid-phase dispersion (MSPD) may be a good alternative for combining extraction and purification procedures, thereby reducing the indicated limitations. Objective Applying an MSPD extraction procedure coupled to high-performance liquid chromatography diode-array detection (HPLC/DAD) as an alternative methodology to evaluate isoflavone profiles. Methods Isoflavone profiles were determined for the leaves of nine species of Medicago in the late flower phenological stage (one or more nodes with 50% open flowers, no seed pods). Extraction was performed following MSPD, and isoflavone profiles were characterised using HPLC/DAD. The quantified amounts were compared with previous results in different species commonly recognised as good sources of isoflavones. Results Formononetin was the major isoflavone in most species, except M. polymorpha and M. truncatula. The isoflavone amounts were significantly different among the assayed species, with M. orbicularis and M. arabica as the major isoflavone sources, while M. rigidula presented the lowest contents. Furthermore, the detected differences allow electing the best species as a primary source of a specific isoflavone. Conclusion The MSPD allowed good extraction efficiency, reproducibility and recovery. Some of the species showed relevant isoflavone contents, even when compared with acknowledged plant sources such as soy or red clover. To the best of our knowledge the results presented are reported for the first time in these species. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 7 August 2014 | 10:59 am


Identification of Phenanthrene Derivatives in Aerides rosea (Orchidaceae) Using the Combined Systems HPLC–ESI–HRMS/MS and HPLC–DAD–MS–SPE–UV–NMR

Introduction In our continued efforts to contribute to the general knowledge on the chemical diversity of orchids, we have decided to focus our investigations on the Aeridinae subtribe. Following our previous phytochemical study of Vanda coerulea, which has led to the identification of phenanthrene derivatives, a closely related species, Aerides rosea Lodd. ex Lindl. & Paxton, was chosen for investigation. Objective To identify new secondary metabolites, and to avoid isolation of those already known, by means of the combined systems HPLC–DAD(diode-array detector) with high-resolution tandem mass spectrometry (HRMS/MS) and HPLC–DAD–MS–SPE(solid-phase extraction)–UV–NMR. Methods A dereplication strategy was developed using a HPLC–DAD–HRMS/MS targeted method and applied to fractions from A. rosea stem extract. Characterisation of unknown minor compounds was then performed using the combined HPLC–DAD–MS–SPE–UV–NMR system. Results The dereplication method allowed the characterisation of four compounds (gigantol, imbricatin, methoxycoelonin and coelonin), previously isolated from Vanda coerulea stem extract. The analyses of two fractions permitted the identification of five additional minor constituents including one phenanthropyran, two phenanthrene and two dihydrophenanthrene derivatives. The full set of NMR data of each compound was obtained from microgram quantities. Conclusion Nine secondary metabolites were characterised in A. rosea stems, utilising HPLC systems combined with high-resolution analytical systems. Two of them are newly described phenanthrene derivatives: aerosanthrene (5-methoxyphenanthrene-2,3,7-triol) and aerosin (3-methoxy-9,10-dihydro-2,5,7-phenanthrenetriol). Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 6 August 2014 | 2:40 pm


Qualitative and Spatial Metabolite Profiling of Lichens by a LC–MS Approach Combined With Optimised Extraction

Introduction Lichens are self-sustaining partnerships comprising fungi as shape-forming partners for their enclosed symbiotic algae. They produce a tremendous diversity of metabolites (1050 metabolites described so far). Objectives A comparison of metabolic profiles in nine lichen species belonging to three genera (Lichina, Collema and Roccella) by using an optimised extraction protocol, determination of the fragmentation pathway and the in situ localisation for major compounds in Roccella species. Methods Chemical analysis was performed using a complementary study combining a Taguchi experimental design with qualitative analysis by high-performance liquid chromatography coupled with mass spectrometry techniques. Results Optimal conditions to obtain the best total extraction yield were determined as follows: mortar grinding to a fine powder, two successive extractions, solid:liquid ratio (2:60) and 700 rpm stirring. Qualitative analysis of the metabolite profiling of these nine species extracted with the optimised method was corroborated using MS and MS/MS approaches. Nine main compounds were identified: 1 ?-orcinol, 2 orsellinic acid, 3 putative choline sulphate, 4 roccellic acid, 5 montagnetol, 6 lecanoric acid, 7 erythrin, 8 lepraric acid and 9 acetylportentol, and several other compounds were reported. Identification was performed using the m/z ratio, fragmentation pathway and/or after isolation by NMR analysis. The variation of the metabolite profile in differently organised parts of two Roccella species suggests a specific role of major compounds in developmental stages of this symbiotic association. Conclusion Metabolic profiles represent specific chemical species and depend on the extraction conditions, the kind of the photobiont partner and the in situ localisation of major compounds. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 6 August 2014 | 2:39 pm


Rapid Determination of Oligopeptides and Amino Acids in Soybean Protein Hydrolysates using High-Resolution Mass Spectrometry

Introduction Soybean protein hydrolysates (SPHs), especially oligopeptides, have shown a variety of functional properties, including immunomodulatory and anti-oxidant effects. Soybean protein hydrolysate products have been used as functional ingredients in food, sports nutrition or clinical nutrition. However, the mixture is mostly undefined due to its complex nature, containing peptides and minor amino acids as well as small proteins. Objectives To develop a specific and efficient method for the identification and structural characterisation of oligopeptides in SPHs, and to determine free amino acids in SPHs in the same analytical run, for evaluation of the chemical profile of SPH products. Methods Accurate mass spectrometry (MS) datasets of SPH samples were recorded on a high-performance liquid chromatography (HPLC) tandem high-resolution (HR) MS system. Potential oligopeptides were tentatively characterised based on their elemental compositions and ring double bond equivalent (RDBE) values, as well as HRMS/MS data. The analytical method to determine amino acids was evaluated in terms of linearity, precision, apparent recovery and limits of detection and quantitation. Results In total, 186 oligopeptides spanning the mass range of m/z 200–1500 and three major free amino acids could be determined in SPH samples in a single sample injection. Ninety-nine oligopeptides were tentatively characterised. The sensitive and specific instrumental performances also permitted the determination of 19 amino acids with a limit of quantitation of???0.1??g/mL. Conclusion The HPLC–HRMS technique has proven to be an advantageous tool for the rapid characterisation of oligopeptides and determination of amino acids in soybean protein hydrolysates. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 7 July 2014 | 6:28 am


Box–Behnken Design for Optimum Extraction of Biogenetic Chemicals from P. lanceolata with an Energy Audit (Thermal?×?Microwave?×?Acoustic): A Case Study of HPTLC Determination with Additional Specificity Using On-line/Off-line Coupling with DAD/NIR/ESI-MS

Introduction The genus Pluchea comprises about 80 species distributed worldwide, out of them, only Pluchea lanceolata (DC.) Oliv. & Hiern, is used extensively in the traditional system of India. No chromatographic method is available for its quality. Objectives To perform the energy audit for the extraction of biogenetic pentacyclic triterpene, its acetate and sterol from P. lanceolata utilising organic and four alternative solvents. Additionally to resolve the uncertainty of TLC determination, on-line/off-line coupling with a diode-array detector (DAD), and near-infrared (NIR) and electrospray ionisation (ESI) MS was introduced. Methods The extraction of taraxasterol (Tx), taraxasterol acetate (TxAc) and stigmasterol (St) from P. lanceolata was performed using three energy modes. The effects of different operating parameters were studied for optimum extraction yield using the design of experiments, that is, the central composite design and Box–Behnken design. In addition to the retention factor (Rf) and visible spectral matching, two additional optical spectroscopic techniques, that is, NIR and ESI-MS, were applied for extended specificity. Results The method was developed for Tx, TxAc and St determination using HPTLC at 645 nm. The optimum extraction yield of targeted compounds was found to be higher with organic solvents than eco-friendly surfactants. The pulse ultrasonic assisted extraction (PUAE) has resulted in optimum extraction of compounds comparable to hot extraction. Both NIR and ESI-MS provided extended specificity in determination. Conclusion The 5/1-PUAE was determined to be effective, reproducible, simple and energy efficient for the determination of Tx, TxAc and St in P. lanceolata. The offline coupling of NIR and ESI-MS with HPTLC led to considerable improvement in specificity. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 7 July 2014 | 6:28 am


Aroma Evaluation of Setonojigiku (Chrysanthemum japonense var. debile) by Hydrodistillation and Solvent-assisted Flavour Evaporation

Introduction The Chrysanthemum genus consisting of about 200 species is mainly distributed over the Northern Hemisphere. Despite the pleasant odour of C. japonense var. debile (setonojigiku), no detailed analysis of the aroma-active compounds has been reported using sensory evaluation. Objectives Using a hydrodistillation (HD) and a solvent-assisted flavour evaporation (SAFE) method to obtain the volatile oil from the leaf parts. Methods To clarify odorants contributing to the characteristic aroma-active compounds, the aroma-extract dilution analysis (AEDA) method was performed through gas chromatography olfactometry (GC/O) analysis. In addition, the odour activity value (OAV) was calculated in order to determine the relative contribution of each compound to the aroma-active compounds. Results A total of 42 components by HD oil were identified by GC–MS, whereas 34 components were identified in SAFE oil. Thirteen compounds were identified by GC/O analysis in HD and SAFE oils respectively. Conclusion Each extraction method has its own advantages and disadvantages, and they are generally complementary to each other. On the basis of AEDA, OAV and sensory evaluations, [2.2.1] bicyclic monoterpenes (borneol, bornyl acetate and camphor) and ?-caryophyllene are considered to be the main aroma-active compounds of both extraction methods. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 30 June 2014 | 5:59 pm


A Universal Quantitative 1H Nuclear Magnetic Resonance (qNMR) Method for Assessing the Purity of Dammarane-type Ginsenosides

IntroductionQuantitative 1H-NMR (qNMR) is a well-established method for quantitative analysis and purity tests. Applications have been reported in many areas, such as natural products, foods and beverages, metabolites, pharmaceuticals and agriculture. The characteristics of quantitative estimation without relying on special target reference substances make qNMR especially suitable for purity tests of chemical compounds and natural products. Ginsenosides are a special group of natural products drawing broad attention, and are considered to be the main bioactive principles behind the claims of ginsengs efficacy. The purity of ginsenosides is usually determined by conventional chromatographic methods, although these may not be ideal due to the response of detectors to discriminate between analytes and impurities and the long run times involved. ObjectiveTo establish a qNMR method for purity tests of six dammarane-type ginsenoside standards. MethodsSeveral experimental parameters were optimised for the quantification, including relaxation delay (D1), the transmitter frequency offset (O1P) and power level for pre-saturation (PL9). The method was validated and the purity of the six ginsenoside standards was tested. Also, the results of the qNMR method were further validated by comparison with those of high performance liquid chromatography. ConclusionThe qNMR method was rapid, specific and accurate, thus providing a practical and reliable protocol for the purity analysis of ginsenoside standards. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 9 June 2014 | 8:07 pm


A Rapid Extraction and Analysis Method for the Simultaneous Determination of 26 Bioflavonoids in Portulaca Oleracea L.

IntroductionPortulaca oleracea L. (P. oleracea, purslane) is an edible plant that is widely distributed around the world, and flavonoids are its main bioactive constituents. Therefore, the detection of flavonoids is very important for a better understanding of its pharmacological actions and to monitor the product quality control of P. oleracea. ObjectiveTo develop a rapid method to extract and determine 26 bioflavonoids in P. oleracea, based on microwave extraction (MWE) and triple quadrupole-linear ion trap mass spectrometry. MethodsThe optimal conditions of MWE for the extraction of flavonoids from P. oleracea involved the use of methanol as the extraction solvent, a microwave power of 300 W, an extraction time of 450 s, and a solvent-to-solid ratio of 30 mL/g. The samples were analysed using an ultra-performance liquid chromatograph coupled with a triple quadrupole-linear ion trap mass spectrometer (UPLC–MS/MS) system. ResultsThe calibration curves of all 26 analytes showed good linearity (r ? 0.999) and the intra- and interday precisions and repeatability were all within required limits. The mean recoveries measured at three concentrations were higher than 94.2%, with RSDs lower than 2.94% for the targets. ConclusionThe established MWE/UPLC–MS/MS method is a rapid and effective method for quality evaluation of P. oleracea from different production regions and different harvest periods. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 29 May 2014 | 4:55 pm


Phenolic Compounds Involved in Grafting Incompatibility of Vitis spp: Development and Validation of an Analytical Method for their Quantification

Introduction Graft incompatibility of Vitis spp is an unresolved worldwide problem with important economic consequences. Grafting comprises a complex set of morphological and physiological alterations, in which the phenolic compounds seem to be strongly involved. Therefore, a detailed analysis and recognition of structural phenolic compounds diversity in the two partners of a Vitis graft is of great importance to evaluate their role as markers of graft establishment. Objective To optimise a sample extraction method, and to develop and validate a high-performance liquid chromatography (HPLC) method for the simultaneous determination of phenolic acids and flavonols in the graft union so as to understand their behaviour in the metabolism of the scion–rootstock system, using compatible and incompatible combinations of a Syrah cultivar and two rootstocks (R110 and SO4). Methods Sixty extracts of Vitis grafting tissues were prepared and analysed by HPLC for the qualitative and quantitative determination of their phenolic profile. Results Among the phenolic compounds identified in the samples, one benzoic acid (gallic acid), three cinnamic acids (caffeic acid, ferulic acid and sinapic acid) and two flavonols (catechin and epicatechin) are potentially suitable as markers of graft incompatibility. Conclusion The method developed presents good performance and lends itself readily for application in routine analysis of the phenolic composition of Vitis grafting tissues to distinguish compatible and incompatible combinations in the graft callusing stage. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 29 May 2014 | 4:55 pm


Evaluation of Salacia Species as Anti-diabetic Natural Resources Based on Quantitative Analysis of Eight Sulphonium Constituents: A New Class of ?-Glucosidase Inhibitors

Introduction Stems and roots of Salacia genus plants have been used in Ayurveda as a specific remedy for early stage diabetes. Previous investigations identified four sulphonium sulphates, that is, salacinol (1), kotalanol (3), ponkoranol (5) and salaprinol (7), as the compounds responsible for the anti-diabetic activity. Their desulphonates (2, 4, 6 and 8) were also isolated as active constituents. Two separate quantitative analytical protocols, that is, for 1 and 3 and for 2 and 4, have been developed recently. Objective To: validate the two analytical protocols with respect to all eight sulphoniums; evaluate the quality of a variety of Salacia samples collected in different geographical regions, that is, Thailand, Sri Lanka and India; and determine their distribution in each part of the plant, that is, stems/roots, leaves and fruits. Methods Analyses of four sulphonium sulphates in 32 Salacia extracts were carried out on an Asahipak NH2P-50 column, and those of the corresponding desulphonates were conducted on an Inertsil ODS-3 column. Results Neokotalanol (4) was the major constituent in Salacia samples from Thailand, whereas 1 was the primary constituent in extracts of the stems/roots of plants from Sri Lanka and India. These sulphoniums were only present in trace amounts in leaves and fruits of the plants. Conclusion Two analytical protocols were successfully applied to analyse 32 Salacia samples, and revealed that sulphoniums (1–8) had characteristic distributions due to the plant part and/or due to geographical region. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 9 May 2014 | 5:08 pm


Semi-automated Separation of the Epimeric Dehydropyrrolizidine Alkaloids Lycopsamine and Intermedine: Preparation of their N-oxides and NMR Comparison with Diastereoisomeric Rinderine and Echinatine

Introduction The diversity of structure and, particularly, stereochemical variation of the dehydropyrrolizidine alkaloids can present challenges for analysis and the isolation of pure compounds for the preparation of analytical standards and for toxicology studies. Objective To investigate methods for the separation of gram-scale quantities of the epimeric dehydropyrrolizidine alkaloids lycopsamine and intermedine and to compare their NMR spectroscopic data with those of their heliotridine-based analogues echinatine and rinderine. Methods Lycopsamine and intermedine were extracted, predominantly as their N-oxides and along with their acetylated derivatives, from commercial samples of comfrey (Symphytum officinale) root. Alkaloid enrichment involved liquid–liquid partitioning of the crude methanol extract between dilute aqueous acid and n-butanol, reduction of N-oxides and subsequent continuous liquid–liquid extraction of free base alkaloids into CHCl3. The alkaloid-rich fraction was further subjected to semi-automated flash chromatography using boronated soda glass beads or boronated quartz sand. Results Boronated soda glass beads (or quartz sand) chromatography adapted to a Biotage Isolera Flash Chromatography System enabled large-scale separation (at least up to 1–2 g quantities) of lycopsamine and intermedine. The structures were confirmed using one- and two-dimensional 1H- and 13C-NMR spectroscopy. Examination of the NMR data for lycopsamine, intermedine and their heliotridine-based analogues echinatine and rinderine allowed for some amendments of literature data and provided useful comparisons for determining relative configurations in monoester dehydropyrrolizidine alkaloids. A similar NMR comparison of lycopsamine and intermedine with their N-oxides showed the effects of N-oxidation on some key chemical shifts. A levorotatory shift in specific rotation from +3.29° to ?1.5° was observed for lycopsamine when dissolved in ethanol or methanol respectively. Conclusion A semi-automated flash chromatographic process using boronated soda glass beads was standardised and confirmed as a useful, larger scale preparative approach for separating the epimers lycopsamine and intermedine. The useful NMR correlations to stereochemical arrangements within this specific class of dehydropyrrolizidine alkaloid cannot be confidently extrapolated to other similar dehydropyrrolizidine alkaloids. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Posted on 9 May 2014 | 5:06 pm


Rapid Chemical Characterisation of Stilbenes in the Root Bark of Norway Spruce by Off-line HPLC/DAD–NMR

Introduction Stilbenes are plant secondary metabolites that have shown promising and varied biological activities. Stilbenes are presently actively studied for the exploitation of this primary raw material resource, involving the concept of biorefining. Methods for the rapid discovery of new and known stilbene structures from various plant sources are thus keenly sought. Objective To establish a simple and rapid technique of off-line HPLC with a diode-array detector (DAD) and NMR for the unambiguous structural elucidation of stilbene structures in the root bark of Norway spruce [Picea abies (L.) Karst.]. Material and methods The stilbene containing fraction was extracted from the plant bark with an ethanol:water mixture (95:5, v/v) preceded by defatting of hydrophobic compounds with n-hexane using the accelerated solvent extraction technique. A portion of the ethanol–water soluble extract was hydrolysed with ?-glucosidase to prepare stilbene aglycones. The extracts were further purified and enriched using a polymeric adsorbent. Stilbene-enriched extracts were directly characterised by off-line HPLC/DAD–NMR in conjunction with HPLC/DAD and HPLC/DAD with electrospray ionisation MSn. Results Trans-isorhapontin and trans-astringin were identified as the major, and trans-piceid as a minor, stilbene glucosides of the bark of roots of Picea abies. Not only stilbene glucosides but also the corresponding stilbene aglycones, such as trans-resveratrol, trans-piceatannol and trans-isorhapontigenin, were rapidly identified from the hydrolysed extract. The acquired heteronuclear single-quantum coherence and heteronuclear multiple bond correlation spectra were used to assign the complete carbon NMR chemical shifts of trans-isorhapontin and trans-astringin without the need of acquiring a 13C-NMR spectrum. Conclusion The off-line HPLC/DAD–NMR method is expedient for the unambiguous identication of structurally similar stilbenes in plant extracts. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 28 April 2014 | 2:48 pm


Liquid Chromatography–Mass Spectrometry and Chemometric Analysis of Ricinus communis Extracts for Cultivar Identification

Introduction Seeds of Ricinus communis contain the toxic protein ricin, a 64 kD heterodimeric type II ribosome-inactivating protein that has been used in several high-profile poisoning incidents. The ability to determine which cultivar the toxin was isolated from via an LC–MS method would be of significant use to law enforcement and forensic agencies. Objective To analyse via LC–MS and chemometrics (principal components analysis (PCA), orthogonal partial-least-squares discriminant analysis (OPLS-DA)) extracts of R. communis to identify compounds specific to a particular cultivar. Methods Seeds from eight specimens of six cultivars of R. communis (‘carmencita’, ‘dehradun’, ‘gibsonii’, ‘impala’, ‘sanguineus’ and ‘zanzibariensis’) were extracted using a standard methodology. These extracts were analysed by LC–MS then subjected to chemometric analysis (PCA and OPLS-DA). Identified compounds of importance were subjected to high-resolution Fourier transform (HRFT) MS and MS/MS to elucidate their structures. Results This analysis identified 17 ions as potential cultivar determinators. Through accurate mass measurement and MS/MS, molecular formulae for 13 ions were determined, including two known and 11 new peptides. Conclusion Unique ions in extracts of ‘carmencita’, ‘dehradun’, ‘gibsonii’, ‘impala’ and ‘zanzibariensis’ were identified that would allow an individual cultivar to be distinguished from other cultivars in this study. Although ‘sanguineus’ extracts contained no unique compounds, a unique LC–MS profile would allow for cultivar assignment. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 16 April 2014 | 7:32 am


A Comparative Tissue-specific Metabolite Analysis and Determination of Protodioscin Content in Asparagus Species used in Traditional Chinese Medicine and Ayurveda by use of Laser Microdissection, UHPLC–QTOF/MS and LC–MS/MS

Introduction Asparagus is esteemed in Traditional Chinese Medicine and Ayurveda, and it is commercially one of the most important drugs in the global herbal market. Comparative metabolite profiling of different species would help in determining the similarities and ascertain their validity for being used as substitutes for each other. Laser microdissection (LMD) facilitates identification of metabolites in specific tissues, and thus it can aid in exploration of metabolic pathways in target tissues. Objective To compare tissue-specific metabolites and protodioscin content of Asparagus cochinchinensis (Lour.) Merr. and Asparagus racemosus Willd. used in China and India. Methods Metabolite analysis of laser-dissected tissues was carried out using UHPLC–QTOF/MS and LC–MS/MS. The protodioscin contents were determined and the method was validated as per the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines. Results Metabolite analysis reveals that the velamen tissue, among other tissues such as cortex, vascular bundles and pith, contained maximum components, specifically those belonging to the steroidal saponin class. Although the metabolite profiles were similar, the content of protodioscin was found to be higher in Chinese than Indian species. Conclusion The study provided a suitable methodology for metabolite profiling and protodioscin content determination of Asparagus by use of LMD, UHPLC–QTOF/MS and LC–MS/MS. The similarities in metabolite profiles indicate that Asparagus species from India and China can serve as substitute for each other in various therapeutic and pharmaceutical applications. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 16 April 2014 | 7:32 am


Identification and Quantitation of Phenolic Compounds from the Seed and Pomace of Perilla frutescens Using HPLC/PDA and HPLC–ESI/QTOF/MS/MS

Introduction Perilla frutescens (L.) Britt., an essential traditional Asian crop and Chinese medicine, potentially exerts anti-oxidation effects through its phenolic compounds. These compounds have already been reported in perilla seed, however, little is reported in Perilla pomace, the primary waste during oil production of Perilla seed. Objective To investigate major phenolic compounds in perilla seeds and pomaces in order to check whether the pomace could be an alternative resource to the seed for nutritional and medical purposes. Methods Compounds in extracts of perilla seeds and pomaces were separated by high-performance liquid chromatography and detected by photodiode array, and by electrospray ionisation with quadrupole time-of-flight tandem mass spectrometry. Herb-markers selected by principal components analysis were then quantified in both seeds and pomaces. Moreover, a fingerprinting approach and multiple discriminant analysis were applied to screen the phenolic markers in 22 samples. Results Ten phenols were tentatively identified, among which four (rosmarinic acid, luteolin, apigenin and rosmarinic acid-3-O-glucoside) were selected as herb-markers. Perilla seeds and pomaces showed similar phenol profiles, however, the pomaces contained almost two times the amount of the four herb-markers than the seeds. Conclusion The results indicated perilla pomace is a promising alternative source of phenolic compounds that could be recovered and potentially used as natural anti-oxidants. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 16 April 2014 | 7:32 am


NoSQL Data Model for Semi-automatic Integration of Ethnomedicinal Plant Data from Multiple Sources

Introduction Sharing traditional knowledge with the scientific community could refine scientific approaches to phytochemical investigation and conservation of ethnomedicinal plants. As such, integration of traditional knowledge with scientific data using a single platform for sharing is greatly needed. However, ethnomedicinal data are available in heterogeneous formats, which depend on cultural aspects, survey methodology and focus of the study. Phytochemical and bioassay data are also available from many open sources in various standards and customised formats. Objective To design a flexible data model that could integrate both primary and curated ethnomedicinal plant data from multiple sources. Materials and methods The current model is based on MongoDB, one of the Not only Structured Query Language (NoSQL) databases. Although it does not contain schema, modifications were made so that the model could incorporate both standard and customised ethnomedicinal plant data format from different sources. Results The model presented can integrate both primary and secondary data related to ethnomedicinal plants. Accommodation of disparate data was accomplished by a feature of this database that supported a different set of fields for each document. It also allowed storage of similar data having different properties. Conclusion The model presented is scalable to a highly complex level with continuing maturation of the database, and is applicable for storing, retrieving and sharing ethnomedicinal plant data. It can also serve as a flexible alternative to a relational and normalised database. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 16 April 2014 | 7:32 am


Ultra-performance LC Separation and Quadrupole Time-of-flight MS Identification of Major Alkaloids in Plumula Nelumbinis

Introduction As an essential medicine and tea source in many countries, Plumula Nelumbinis potentially exerts its major biological activities through its alkaloids. However, the activities of Plumula Nelumbinis are not fully understood due to the lack of studies on its chemical components. Objective To establish an ultra-performance liquid chromatography combined with diode-array detector (UPLC/DAD) method, coupled to an electrospray ionisation with quadrupole time-of-flight mass spectrometry (ESI/QTOF/MS) method, for the separation and identification of Plumula Nelumbinis alkaloids. Methods The eluant from an UPLC separation of an ethanol extract of Plumula Nelumbinis was directly infused into an ESI/QTOF/MS system. Both positive and negative ion modes of ESI with low and high collision energy (CE) were used to obtain sufficient MS information. Results Twenty-one alkaloids were tentatively identified based on their chromatographic characteristics, UV spectra, exact mass, MS fragments and literature reports. They consist of six bis-1-benzyltetrahydroisoquinoline, eleven benzyltetrahydroisoquinoline (including two glycoalkaloids and two quaternary ammoniums), two aporphine, one proaporphine and one indole alkaloids. Eleven were identified in Plumula Nelumbinis for the first time and seven were first reported in Nelumbo nucifera Gaertn. Five compounds, namely norcoclaurine-4?-O-glucoside, norcoclaurine-6-O-glucoside, isolotusine, 6-demethyl-4-demethylN-methylcoclaurine and N-norisoliensinine, were characterised and proposed as new compounds. Conclusion The established UPLC/DAD???ESI/QTOF/MS method is efficient for systematic identification of the alkaloids in Plumula Nelumbinis extract. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 15 April 2014 | 1:26 am


Evaluation of Anti-oxidant and Acetylcholinesterase Activity and Identification of Polyphenolics of the Invasive Weed Dittrichia viscosa

Introduction The bioactive metabolites derived from weeds have attracted the interest of the food and pharmaceutical industries due to their health benefits. Objective To evaluate the anti-oxidant and acetylcholinesterase activity of Dittrichia viscosa extracts and characterise the polyphenolic metabolites using the LC coupled with diode-array detection (DAD) and positive mode electrospray ionisation (ESI) MS method with a view to evaluating the exploitation potential of this invasive weed. Materials and methods Roots and aerial parts of D. viscosa were extracted with solvents of increasing polarity and their major polyphenolic metabolites were identified by LC???DAD/ESI(+)/MS. The total phenolic content of the extracts was determined using the Folin–Ciocalteu method, while their anti-oxidant activity was evaluated on the basis of their ability to scavenge the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydrogen peroxide. Thin-layer chromatography was used to screen for acetylcholinesterase inhibitors. Results Stem extracts gave the highest phenolic content, whereas the roots showed the lowest content. Twenty-five polyphenolic constituents of the extracts were tentatively characterised according to their MS and UV spectroscopic data. Among the extracts studied, roots–ethyl acetate and flowers–diethyl ether revealed the highest activity according to the DPPH and chemiluminescence assays respectively. Conclusion The metabolic profile of D. viscosa was studied and the structures of the major polyphenolic metabolites were tentatively assigned based on their MS and UV–vis spectra. The extracts exhibited high levels of anti-oxidant and acetylcholinesterase inhibitory activity and the inhibitors are probably localised mainly in flowers and roots. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 14 April 2014 | 5:01 pm


Optimisation of the Microplate Resazurin Assay for Screening and Bioassay-guided Fractionation of Phytochemical Extracts against Mycobacterium tuberculosis

Introduction Because of increased resistance to current drugs, there is an urgent need to discover new anti-mycobacterial compounds for the development of novel anti-tuberculosis drugs. The microplate resazurin assay (MRA) is commonly used to evaluate natural products and synthetic compounds for anti-mycobacterial activity. However, the assay can be problematic and unreliable when screening methanolic phytochemical extracts. Objective To optimise the MRA for the screening and bioassay-guided fractionation of phytochemical extracts using Mycobacterium tuberculosis H37Ra. Methods The effects of varying assay duration, resazurin solution composition, solvent (dimethyl sulphoxide – DMSO) concentration and type of microtitre plate used on the results and reliability of the MRA were investigated. The optimal bioassay protocol was applied to methanolic extracts of medicinal plants that have been reported to possess anti-mycobacterial activity. Results The variables investigated were found to have significant effects on the results obtained with the MRA. A standardised procedure that can reliably quantify anti-mycobacterial activity of phytochemical extracts in as little as 48 h was identified. The optimised MRA uses 2% aqueous DMSO, with an indicator solution of 62.5 µg/mL resazurin in 5% aqueous Tween 80 over 96 h incubation. Conclusion The study has identified an optimal procedure for the MRA when used with M. tuberculosis H37Ra that gives rapid, reliable and consistent results. The assay procedure has been used successfully for the screening and bioassay-guided fractionation of anti-mycobacterial compounds from methanol extracts of Canadian medicinal plants. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 14 April 2014 | 5:01 pm


Piper betle Leaves: Profiling Phenolic Compounds by HPLC/DAD–ESI/MSn and Anti-cholinesterase Activity

Introduction Piper betle L. is a widely distributed plant in the tropical and subtropical regions, its leaves being largely consumed as a masticator and mouth freshener. Objective The purposes of this work were to characterise the phenolic profile of this species and to improve knowledge of its anti-cholinesterase properties. Methods The phenolic composition of P. betle leaf aqueous and ethanol extracts was characterised by HPLC coupled with a diode-array detector and combined with electrospray ionisation tandem MS, and in vitro cholinesterase inhibitory capacity of both extracts was assessed by spectrophotometric microassays. The effect on neuronal cells (SH-SY5Y) viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and lactate dehydrogenase leakage. Results Twelve phenolic compounds, comprising a phenylpropanoid, five cinnamoyl and six flavonoids derivatives were identified in P. betle leaves. Hydroxychavicol was the major compound in both extracts; however, the aqueous extract presented a greater diversity of compounds. Both extracts showed strong activity against both acetyl- and butyrylcholinesterase, which can be due, at least partially, to the phenolic composition. Furthermore, the aqueous extract proved to be cytotoxic to human neuroblastoma cells at concentrations higher than 500?µg/mL. Conclusion The results suggest that the consumption of P. betle leaves as an infusion can have a positive impact in the prevention and treatment of neurodegenerative diseases. Apigenin and luteolin derivatives are reported for the first time in this species. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 14 April 2014 | 5:01 pm


Microplate Quantification of Total Phenolic Content from Plant Extracts Obtained by Conventional and Ultrasound Methods

Introduction There is increasing interest in phenolic compounds around the world because of their potential positive impact on human health. Phenolic compounds are largely found in fruits and vegetables. Extraction of phenolic compounds is a very important step in their recovery. The newly developed technique of ultrasound-assisted extraction (UAE) appears to be an advantageous alternative compared with conventional techniques, because it is simple and environmental friendly. The potential of UAE needs to be evaluated in each plant in order to demonstrate its efficiency. Objective The objective of the present study was to compare a conventional method and UAE on the extraction efficiency of phenolic compounds from Jatropha dioica, Fluorensia cernua, Turnera diffusa and Eucalyptus camaldulensis plants and evaluate the in vitro anti-oxidant potential. Methods Validation of the new method was carried out using mixed-model methodology and regression analysis. Feasibility of this new method was shown and applied using several plants extracts obtained by different extraction methods from semi-arid Mexican plants, which were characterised by high levels of polyphenols. Additionally, the anti-oxidant potential of these extracts was determined by 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Results Results showed that the new microplate method can be used to determine total phenolic content in plant extracts. Additionally, an alternative extraction method by ultrasound was less efficient compared with the conventional method. Conclusion The tested plants are good candidates to obtain nutraceuticals and functional food ingredients. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 1 April 2014 | 4:20 pm


Near-infrared Reflectance Spectroscopy as a Rapid and Non-destructive Analysis Tool for Curcuminoids in Turmeric

Introduction Turmeric has been widely used in curry powders as the main spice. Conventional chemical analysis such as high-performance liquid chromatography (HPLC) may take several hours to extract curcuminoids and prepare samples in many turmeric processing industries. Objective This study was conducted to evaluate curcuminoids in turmeric powder using near-infrared reflectance spectroscopy (NIRS). Methods All spectral acquisition ranged from 1100 to 2500 nm and a chemometrics analysis using partial least-squares (PLS) regression was performed to quantify the contents of individual curcuminoids. The HPLC was carried out (n = 129) to develop a PLS model based on the reference values. Results High correlation coefficient (R2 > 0.93) and low standard error of cross-validation (SECV < 0.20 g/100 g) and standard error of prediction (SEP < 0.13 g/100 g) values were obtained for precision and accuracy. In addition, the ratio of prediction to deviation (RPD > 2.65) values was also calculated. Conclusion Our results indicate that NIRS could be utilised as a control procedure or as an alternative rapid and effective quantification method. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 1 April 2014 | 4:03 pm


Monodimensional (GC–FID and GC–MS) and Comprehensive Two-dimensional Gas Chromatography for the Assessment of Volatiles and Fatty Acids from Ruta chalepensis Aerial Parts

Introduction Ruta chalepensis L. (Rutaceae) is widespread in the Mediterranean area. This plant has a solid tradition in ethnomedicine because of its various biological activities. Based on previous reports, the main volatile constituents of R. chalepensis are 2-undecanone and 2-nonanone, but most are still unknown, particularly fatty acid composition. Objective To exhaustively characterise the chemical composition of the aerial parts from R. chalepensis plants collected from the wild in Sicily, within a project aiming at the evaluation and characterisation of medicinal plants from the Mediterranean flora. The study was directed toward the determination of volatiles and fatty acids in samples of R. chalepensis obtained from different aerial plant parts and from plants harvested at different times. Methods GC with flame ionisation detection, GC–MS and two-dimensional gas chromatography (GC?×?GC) advanced techniques, with support of dedicated mass spectral databases provided with retention index (RI) information, were applied to determine both volatiles and fatty acids. Samples were extracted by hydrodistillation and underwent methylic transesterification in order to be transformed into the correspondent fatty acid methyl esters (FAMEs). Results The monodimensional analysis by GC–MS with RI confirmed that 2-nonanone and 2-undecanone are the predominant components in all the plant parts, followed by esters and monoterpenes. A different distribution was observed of the main compounds in the various plant parts depending on the life cycle of the plant (vegetative or reproductive stage). The multidimensional GC?×?GC analysis allowed for a complete screening of the fatty acids. About 65% of the total were polyunsaturated fatty acids (PUFA), followed by 30% of saturated fatty acids (SFA). Conclusion A detailed GC volatile fingerprint of R. chalepensis flowers, leaves, fruits and stems was established, highlighting the compositional differences depending on plant organs and life cycle. The results indicated R. chalepensis as a good source of fatty acids from the w3 and w6 series. In both essential oil and lipidic extract, many compounds were determined for the first time. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 1 April 2014 | 4:03 pm


Rapid Identification of Polymethoxylated Flavonoids in Traditional Chinese Medicines with a Practical Strategy of Stepwise Mass Defect Filtering Coupled to Diagnostic Product Ions Analysis based on a Hybrid LTQ-Orbitrap Mass Spectrometer

Introduction The methodology of stepwise mass defect filtering (MDF) approach coupled to diagnostic product ions (DPIs) analysis on a hybrid linear trap quadrupole (LTQ)/orbitrap mass spectrometer was the first to be established to screen and identify structural analogues from complex herbal extracts. Objective To develop an analytical methodology that could be adopted to screen and identify structural analogues in traditional Chinese medicines (TCMs) rapidly and accurately. Methods Taking polymethoxylated flavonoids (PMFs) in the leaves of Citrus reticulata Blanco as an example, high-resolution mass data were acquired by high-performance liquid chromatography (HPLC) coupled with a LTQ/orbitrap mass spectrometer. The stepwise MDF with multiple mass defect windows or mass windows enabled the original data to be analysed much faster and more accurately by reducing the potential interferences of matrix ions. Additionally, analysis of DPIs could provide a criterion to classify the target constituents detected into certain chemical families. Results In total, 81 PMFs, including 50 polymethoxyflavones and 31 polymethoxyflavanones or polymethoxychalcones, were screened and identified from the original data and preliminarily identified. Conclusion The analytical methodology developed could be used as a rapid, effective technique to screen and identify compounds from TCM extracts and other organic matter mixtures with compounds that can also be classified into families based on the common carbon skeletons. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 5 March 2014 | 10:41 am


A Simple Semi-preparative Reversed-phase HPLC/PDA Method for Separation and Quantification of Glycyrrhizin in Nine Samples of Glycyrrhiza glabra Root Collected from Different Geographical Origins

Introduction Glycyrrhiza glabra L. (Fabaceae), commonly known as ‘liquorice’, is one of the most popular ingredients in several traditional herbal medicinal preparations, and glycyrrhizin is the major glycoside present in this plant. The content of glycyrrhizin may vary among G. glabra samples collected from various geographical origins, which may affect the therapeutic efficacy. Thus, quantification of glycyrrhizin in G. glabra samples is important. Objective To develop and validate a simple semi-preparative reversed-phase HPLC with photodiode array (PDA) method for separation and quantification of glycyrrhizin in nine samples of G. glabra root collected from various geographical origins. Methods Dried and ground root of G. glabra was Soxhlet-extracted sequentially with n-hexane and methanol (MeOH). The separation and quantification of glycyrrhizin was achieved on a C18 reversed-phase semi-preparative column using a gradient mobile phase, 30–100% solvent B in solvent A in 30 min (solvent A: 0.1% v/v trifluoroacetic acid (TFA) in water and solvent B: 0.1% v/v of TFA in MeOH), at a flow rate of 3.00 mL/min and UV detection at 254 nm. Results A simple semi-preparative reversed-phase HPLC/PDA method allowing clear separation and quantification of glycyrrhizin content in nine samples has been validated in terms of linearity, selectivity, limits of detection, precision, accuracy and detection. Concentration levels of glycyrrhizin were between 0.177 and 0.688% w/w of dry materials. Conclusion This method is precise, less time consuming and more cost effective, and can be used for the quality control of any G. glabra sample with regard to its glycyrrhizin contents. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 3 March 2014 | 11:13 am


Quantitative Analysis of Boeravinones in the Roots of Boerhaavia Diffusa by UPLC/PDA

Introduction Boerhaavia diffusa is a perennial herb belonging to Nyctaginaceae. Various classes of chemical constituents such as phenolics (boeravinones), terpenoids and organic acids have been reported in B. diffusa roots. As boeravinones have been proposed as putative active constituents for the anti-cancer, spasmolytic and anti-inflammatory activities exhibited by B. diffusa extracts, it is worthwhile developing and validating an ultra-performance liquid chromatography (UPLC) method for analysis of boeravinones in B. diffusa roots. Objective To develop and validate a simple, accurate, robust and rapid UPLC analytical method for quality control of B. diffusa roots. Methods Samples for analysis were prepared by refluxing powdered root material with methanol for 2 h. The extracts were concentrated, dried and stored at –20°C until their use. A UPLC with photodiode array (PDA) method was developed and validated for the quantification of boeravinones in the roots of B. diffusa. The separation of boeravinones was achieved using a BEH Shield C18-column (2.1 × 100 mm, 1.7 µm) with gradient elution of methanol and water (0.1% acetic acid), at a flow rate of 0.4 mL/min and detection was carried out at ?max 273 nm. Results The UPLC method developed showed good linearity (r2???0.9999), accuracy and precision. Conclusion The UPLC method developed provided a selective, sensitive and rapid analytical method for the quantification of boeravinones in B. diffusa roots. All the validation parameters were found to be within the permissible limits as per International Conference on Harmonisation guidelines. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 21 February 2014 | 9:53 am


Tentative Characterisation of Iridoids, Phenylethanoid Glycosides and Flavonoid Derivatives from Globularia alypum L. (Globulariaceae) Leaves by LC-ESI-QTOF-MS

Introduction Globularia alypum L., belonging to the Globulariaceae family, is a perennial wild shrub found throughout the Mediterranean area, Europe, and Africa. This plant is widely used to treat many diseases, but no previous work on the phytochemical composition of the Algerian G. alypum species has yet been reported. Objective To investigate the phytoconstituents of the methanolic extract of G. alypum using an LC-ESI-QTOF-MS method. Methods Ground air-dried leaves of G. alypum were macerated with methanol at room temperature for 24?h. The supernatant was filtered and concentrated to dryness under reduced pressure in a rotary evaporator, and extracts were recovered with methanol and filtered. Afterwards, the G. alypum extract was injected into the LC-ESI-QTOF-MS system. Results The combined LC–MS/MS led to the tentative characterisation of 63 phytochemicals. In this work, a large number of compounds have been characterised in the leaf-extract analysis of this plant. Among others, 24 iridoids and secoiridoids were found, of which nine compounds have not previously been recorded in G. alypum. Also, nine unusual phenylethanoid glycosides were characterised for the first time in this species. Conclusion The method used has proved to be a valued tool for the characterisation of a wide range of compounds from G. alypum leaves. This work constitutes a detailed investigation of the chemical composition of G. alypum leaves, which are widely used in different traditional systems of medicine. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 12 February 2014 | 11:30 am





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