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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:

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

Extraction for Metabolomics: Access to The Metabolome

Introduction The value of information obtained from a metabolomic study depends on how much of the metabolome is present in analysed samples. Thus, only a comprehensive and reproducible extraction method will provide reliable data because the metabolites that will be measured are those that were extracted and all conclusions will be built around this information. Objective To discuss the efficiency and reliability of available sample pre-treatment methods and their application in different fields of metabolomics. Methods The review has three sections: the first deals with pre-extraction techniques, the second discusses the choice of extraction solvents and their main features and the third includes a brief description of the most used extraction techniques: microwave-assisted extraction, solid-phase extraction, supercritical fluid extraction, Soxhlet and a new method developed in our laboratory – the comprehensive extraction method. Results Examination of over 200 studies showed that sample collection, homogenisation, grinding and storage could affect the yield and reproducibility of results. They also revealed that apart from the solvent used for extraction, the extraction techniques have a decisive role on the metabolites available for analysis. Conclusion It is essential to evaluate efficacy and reproducibility of sample pre-treatment as a first step to ensure the reliability of a metabolomic study. Among the reviewed methods, the comprehensive extraction method appears to provide a promising approach for extracting diverse types of metabolites. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 13 February 2014 | 7:22 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

One-step Separation and Purification of Two Chromones and One Pyrone from Aloe barbadensis Miller: a Comparison Between Reversed-phase Flash Chromatography and High-speed Counter Current Chromatography

Introduction Chromones and pyrones are the major secondary metabolites of Aloe barbadensis Miller. As they are minor components of the plant, an efficient purification procedure for them is of great importance for promoting their pharmacological studies. Objective To develop efficient methods for one-step separation and purification of two chromones (5-((S)-2?-oxo-4?-hydroxypentyl)-2-hydroxymethylchromone (1) and 5-((4E)-2?-oxo-pentenyl)-2-hydroxymethylchromone (3)) and one pyrone (aloenin aglycone (2)) from A. barbadensis via reversed-phase flash chromatography (RP-FC) and high-speed counter current chromatography (HSCCC). Methods The RP-FC separation was performed using methanol:water (26:74, v/v) as the mobile phase at a flow rate of 20?mL/min. A solvent system composed of dichloromethane:methanol:water (3:1.5:1, v/v/v) was used for the HSCCC separation, at a flow rate of 2.0?mL/min. Results A one-step RP-FC operation within 110?min was successfully used for the purification of compounds 1 (27.9?mg, 96.5%), 2 (32.4?mg, 98.2%) and 3 (4.1?mg, 99.0%) from 129?mg of crude sample, and a one-step HSCCC separation within 95?min was successfully implemented for the purification of compounds 1 (31.1?mg, 97.6%), 2 (35.8?mg, 96.7%) and 3 (2.7?mg, 98.1%) from 134?mg of crude sample. Conclusion The developed procedures were efficient, with low cost and high yield, which would afford sufficient amounts of high-purity compounds for chromatographic purposes and pharmacological activity screening. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 5 February 2014 | 6:26 am

Thin-layer Chromatographic Identification of Chinese Propolis Using Chemometric Fingerprinting

Introduction Poplar tree gum has a similar chemical composition and appearance to Chinese propolis (bee glue) and has been widely used as a counterfeit propolis because Chinese propolis is typically the poplar-type propolis, the chemical composition of which is determined mainly by the resin of poplar trees. The discrimination of Chinese propolis from poplar tree gum is a challenging task. Objective To develop a rapid thin-layer chromatographic (TLC) identification method using chemometric fingerprinting to discriminate Chinese propolis from poplar tree gum. Methods A new TLC method using a combination of ammonia and hydrogen peroxide vapours as the visualisation reagent was developed to characterise the chemical profile of Chinese propolis. Three separate people performed TLC on eight Chinese propolis samples and three poplar tree gum samples of varying origins. Five chemometric methods, including similarity analysis, hierarchical clustering, k-means clustering, neural network and support vector machine, were compared for use in classifying the samples based on their densitograms obtained from the TLC chromatograms via image analysis. Results Hierarchical clustering, neural network and support vector machine analyses achieved a correct classification rate of 100% in classifying the samples. A strategy for TLC identification of Chinese propolis using chemometric fingerprinting was proposed and it provided accurate sample classification. Conclusion The study has shown that the TLC identification method using chemometric fingerprinting is a rapid, low-cost method for the discrimination of Chinese propolis from poplar tree gum and may be used for the quality control of Chinese propolis. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 4 February 2014 | 1:30 pm

Sample Preparation Issues in NMR-based Plant Metabolomics: Optimisation for Vitis Wood Samples

Introduction Nuclear magnetic resonance (NMR) is one of the most commonly used analytical techniques in plant metabolomics. Although this technique is very reproducible and simple to implement, sample preparation procedures have a great impact on the quality of the metabolomics data. Objective Investigation of different sample preparation methods and establishment of an optimised protocol for untargeted NMR-based metabolomics of Vitis vinifera L. wood samples. Methods Wood samples from two different cultivars of V. vinifera with well-defined phenotypes (Gamaret and 2091) were selected as reference materials. Different extraction solvents (successively, dichloromethane, methanol and water, as well as ethyl acetate and 7:3 methanol-water (v/v)) and deuterated solvents (methanol-d4, 7:3 chloroform-d-methanol-d4 (v/v), dimethylsulphoxide-d6 and 9:1 dimethylsulphoxide-d6-water-d2 (v/v)) were evaluated for NMR acquisition, and the spectral quality was compared. The optimal extract concentration, chemical shift stability and peak area repeatability were also investigated. Results Ethyl acetate was found to be the most satisfactory solvent for the extraction of all representative chemical classes of secondary metabolites in V. vinifera wood. The optimal concentration of dried extract was 10 mg/mL and 7:3 chloroform-d-methanol-d4 (v/v) was the most suitable solvent system for NMR analysis. Multivariate data analysis was used to estimate the biological variation and clustering between different cultivars. Conclusion Close attention should be paid to all required procedures before NMR analysis, especially to the selection of an extraction solvent and a deuterated solvent system to perform an extensive metabolomic survey of the specific matrix. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 4 February 2014 | 1:29 pm

Simultaneous Characterisation of Fifty Coumarins from the Roots of Angelica dahurica by Off-line Two-dimensional High-performance Liquid Chromatography Coupled with Electrospray Ionisation Tandem Mass Spectrometry

Introduction The root of Angelica dahurica is a traditional Chinese medicine that used for the treatment of headache, toothache, abscess, furunculosis and acne. Coumarins were the major bioactive constituents of A. dahurica, hence it is worthwhile developing a method to simultaneously characterise them, especially those in trace amounts. Objective To develop an efficient method for the simultaneous characterisation of coumarins in A. dahurica. Methods A method using off-line two-dimensional high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (off-line 2D-HPLC–ESI/MSn) was developed. Results In total 50 coumarins, including 32 linear furanocoumarins, 16 bifuranocoumarins and two non-furanocoumarins, were identified from the roots of A. dahurica. The possible MS fragmentations of these coumarins are also proposed. Conclusion The method described here allows rapid and convenient identification of the coumarins in A. dahurica, and may be applied to other herbal medicines containing linear furanocoumarins. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 30 January 2014 | 3:08 pm

An Easy, Convenient Cell and Tissue Extraction Protocol for Nuclear Magnetic Resonance Metabolomics

Introduction As a complement to the classic metabolomics biofluid studies, the visualisation of the metabolites contained in cells or tissues could be a very powerful tool to understand how the local metabolism and biochemical pathways could be affected by external or internal stimuli or pathologies. Therefore, extraction and/or lysis is necessary to obtain samples adapted for use with the current analytical tools (liquid NMR and MS). These extraction or lysis work-ups are often the most labour-intensive and rate-limiting steps in metabolomics, as they require accuracy and repeatability as well as robustness. Many of the procedures described in the literature appear to be very time-consuming and not easily amenable to automation. Objective To find a fast, simplified procedure that allows release of the metabolites from cells and tissues in a way that is compatible with NMR analysis. Methods We assessed the use of sonication to disrupt cell membranes or tissue structures. Both a vibrating probe and an automated bath sonicator were explored. Results The application of sonication as the disruption procedure led to reproducible NMR spectral data compatible with metabolomics studies. This method requires only a small biological tissue or cell sample, and a rapid, reduced work-up was applied before analysis. The spectral patterns obtained are comparable with previous, well-described extraction protocols. Conclusion The rapidity and the simplicity of this approach could represent a suitable alternative to the other protocols. Additionally, this approach could be favourable for high- throughput applications in intracellular and intratissular metabolite measurements. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 23 January 2014 | 11:33 am

Tandem Solid-Phase Extraction Followed by HPLC–ESI/QTOF/MS/MS for Rapid Screening and Structural Identification of Trace Diterpenoids in Flowers of Rhododendron molle

Introduction ‘Naoyanghua’, composed of the flowers of Rhododendron molle G. Don, is a traditional Chinese medicine that is widely known for its toxicity. Grayanane-type diterpenoids are the main active ingredients in R. molle, as well as possibly their toxicity: they are, however, difficult to isolate and analyse using common chromatographic methods, due to their small amounts and absence of conjugated groups, such as phenyl and ?, ?-unsaturated ketone. Objective To establish a highly sensitive, selective and reliable method for the qualitative evaluation of trace diterpenoids in the flowers of R. molle by using tandem solid-phase extraction followed by high-performance liquid chromatography with electrospray ionisation quadrupole-time-of-flight mass spectrometry (HPLC–ESI/QTOF/MS/MS). Methods Tandem solid phase extraction (SPE) was undertaken using a polyamide cartridge and a C18E cartridge in succession to enrich the trace diterpenoids. HPLC–ESI/QTOF/MS/MS was used to determine the fragmentation patterns of diterpenoids and to tentatively characterise their fragmentation pathways. Results HPLC–ESI/QTOF/MS/MS detected a total of 14 diterpenoids, eight of which were identified by comparison with literature sources and six based on fragmentation analysis. Among the latter six, rhodojaponin VI-3-glucoside was tentatively identified as a new diterpenoid glycoside and rhodojaponin VII, rhodojaponin IV and rhodojaponin I were reported from R. molle for the first time. Conclusion By qualitative research of diterpenoids in this plant by HPLC–ESI/QTOF/MS/MS, a reliable methodology for the analysis of these active constituents of R. molle was established for the first time. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 23 January 2014 | 11:33 am

Rapid Separation of Free Fatty Acids in Vegetable Oils by Capillary Zone Electrophoresis

Introduction Olive oil is a very important product to human health since it inhibits formation of free radicals, tumour growth, lesions and inflammatory substances. High concentrations of free fatty acids in olive oils results in lipid deterioration due to oxidative or hydrolytic rancidity. Objective To optimise an alternative capillary zone electrophoresis methodology, under ultraviolet indirect detection and to determine free fatty acids in edible vegetable oils without derivatisation steps in sample preparation. Methods The condition used consisted of 15?mm NaH2PO4–Na2HPO4 at pH ~6.86, 4.0?mm of sodium dodecybenzenesulphonate, 8.3?mm of Brij 35®, 45% v/v of acetonitrile and 2.1% of 1-octanol, injection at 12.0?mbar of pressure for 4?s, +19?kV of applied voltage and indirect detection at 224?nm, within an analysis time of 11?min. Results The capillary zone electrophoresis method was successfully applied to determination of free fatty acids in extra virgin olive oil, virgin olive oil and soybean oil samples. The comparison with the official volumetric titration method showed no significant difference within the 95% confidence interval. Conclusion The main advantage to the proposed method is the possibility to observe the individual amount of the free fatty acids, which would be useful for researchers interested in studying the effect of the free fatty acids profile on oxidative process in food. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 23 January 2014 | 11:32 am

Sensitive and Selective Determination of Phenolic Compounds from Aromatic Plants Using an Electrochemical Detection Coupled with HPLC Method

Introduction Phenolic compounds contained in essential oils from plants are responsible for their anti-oxidant capacity. The natural extract from each aromatic plant is characterised by a typical ratio of phenolic components, so each one of the essential oils shows different properties. Objective The development of a simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method for the determination of phenolic compounds from aromatic plants using spectrophotometric detection with a diode-array and electrochemical detection with amperometric and coulometric detectors. Methods Chromatographic conditions are optimised to separate vanillin, eugenol, thymol and carvacrol using spectrophotometric detection. Acetonitrile and methanol are studied as mobile-phase organic modifiers. The hydrodynamic curves are obtained for both electrochemical detection modes and the principal values of merit are calculated. The proposed methodology is applied to determine the four analytes in real samples. Results The shortest elution times and the highest electrochemical signals are achieved using 65% methanol solution in 0.1 mol/L acetic acid–acetate buffer as the mobile phase. Potential values of 0.925 V for amperometric detection and 0.500 V for coulometric detection are chosen as working potentials. The limits of detection (LOD) for the compounds studied ranged between 9.7–17 µg/L and 0.81–3.1 µg/L in amperometric and coulometric detection modes, respectively. In general, the obtained LODs are better than those previously reported. Conclusion The low LODs obtained using coulometric detection make this methodology very competitive and adequate for quality control of these phenolic compounds in comparison with others, such as GC–MS, that are more expensive and complicated to use than the RP-HPLC method with coulometric detection. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 23 January 2014 | 11:32 am

Chemometric Resolution Approaches in Characterisation of Volatile Constituents in Plantago ovata Seeds using Gas Chromatography–Mass Spectrometry: Methodology and Performance Assessment

Introduction Comprehensive chemical profiling of herbal medicines (HMs) is a major challenge in chemical characterisation of source materials. Many analytical platforms such as gas chromatography–mass spectrometry (GC–MS) have been applied to the characterisation. However, the great complexity of analytical results has been an obstacle. Chemometric resolution methods as a supplementary tool for data processing are proposed for solving this problem. Objective To develop and demonstrate the ability of chemometric techniques in the characterisation of volatile components in herbal medicines. Methods The volatile components of Plantago ovata were extracted using a solvent extraction method. GC–MS analysis were performed using an Agilent HP-6890 gas chromatograph equipped with a HP-5MS capillary, interfaced with an Agilent HP- 5973 mass selective detector. Resolved spectra were identified by matching against the standard mass spectral database of the National Institute of Standards and Technology (NIST). Results Results of this study show that the 71 constituents that are qualitatively recognised represent 94.53% of the total relative content of constituents from Plantago ovata oil, whereas without applying the chemometric methods only 51 constituents were recognised by direct searching utilising a mass database. In addition the presence of valuable components such as thymol, 2,4-decadienal, linoleic acid and oleic acid in Plantago ovata oil has been demonstrated. Conclusion GC–MS combined with chemometric resolution methods, such as multivariate curve resolution–alternating least squares (MCR–ALS), will provide a reliable means for rapid and accurate analyses of unknown complicated practical systems. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 23 January 2014 | 11:31 am

HPLC/UV Analysis of Polyacetylenes, Phenylpropanoid and Pyrrolidine Alkaloids in Medicinally Used Codonopsis Species

Introduction Codonopsis Radix is commonly used as a tonic in traditional Chinese medicine. However, there is no suitable method to assess the quality of Codonopsis Radix based on multiple components having potential bioactivities. Objective To establish a HPLC/UV method for simultaneous quantitation of polyacetylenes (lobetyol, lobetyolin, lobetyolinin, cordifolioidyne B), phenylpropanoid (tangshenoside I) and pyrrolidine alkaloids (codonopyrrolidiums A, B) in Codonopsis Radix. Methods Large-scale methanol extraction of Codonopsis Radix, followed by chromatographic separation, provided the seven analytes for quantitation standards. Ultrasound-assisted methanol extracts of samples were analysed using reversed phase, gradient elution HPLC monitored at 215?nm. Results The method developed allowed efficient separation of the seven compounds and the detection and quantitation limits of the seven analytes were 0.10–0.32?µg/mL and 0.35–1.07?µg/mL, respectively. All calibration curves showed good linearities (r?>?0.9993) within the test ranges. Intraday and interday precisions were good with RSD?<?2.84%. The recoveries of all analytes ranged from 95.8 to 104.7%. Conclusion HPLC/UV is an efficient and accurate method of analysis for simultaneous quantitation of seven components in Codonopsis Radix. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 23 January 2014 | 11:31 am

Metabolite Profiling of Leek (Allium porrum L) Cultivars by 1H NMR and HPLC–MS

Introduction Leek (Allium ampeloprasum var. porrum) is consumed as a vegetable throughout the world. However, little is known about the metabolites of leek cultivars, especially those with potentially important beneficial properties for human health. Objective We provide new information for the overall metabolite composition of several leek cultivars grown in Europe by using HPLC–MS and 1H NMR. Methods The use of a novel CTLS/NMR (constrained total-line-shape nuclear magnetic resonance) approach was found to be capable of reliable quantification, even with overlapping metabolite signals in the 1H NMR of plant metabolites. Additionally, a new application for leek flavonoids was optimised for HPLC–MS. Results The total concentration of carbohydrates (glucose, fructose, kestose/nystose and sucrose) and nine amino acids varied by fourfold in leek juice from different cultivars, while the total concentrations of four organic acids were similar in all cultivars. All the quantified flavonols were kaempferol derivatives or quercetin derivatives and threefold differences in flavonol concentrations were detected between cultivars. Conclusion In this study, various phytochemical profiles were determined for several leek cultivars by 1H NMR spectroscopy with CTLS combined with HPLC–MS. The wide variation in bioactive compounds among commercial leek cultivars offers promising opportunities for breeders to raise the levels of important biochemical compounds in leek breeding lines, and also provides some objective measure for quality assurance for the leek industry. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 10 January 2014 | 11:34 am

Determination of C-glucosidic Ellagitannins in Lythri salicariaeherba by Ultra-High Performance Liquid Chromatography Coupled with Charged Aerosol Detector: Method Development and Validation

Introduction Lythri salicariaeherba is a pharmacopoeial plant material used by patients in the form of infusions in the treatment of acute diarrhoea. According to its pharmacopoeial monograph it is standardised for total tannin content, which should be not less than 5.0% using pyrogallol as a standard. Previous studies have shown that aqueous extracts from Lythri herba contain mainly ellagitannins among which vescalagin, castalagin and salicarinins A and B are dominating constituents. Objective To develop and validate an efficient UHPLC coupled with a charged aerosol detector (CAD) method for quantification of four major ellagitannins in Lythri salicariaeherba and in one commercial preparation. Methods Extraction conditions of ellagitannins from plant material were optimised. The relative response factors for vescalagin, castalagin and salicarinins A and B using gallic acid as an external standard were determined for the CAD detector. Then, a UHPLC method for quantification of ellagitannins was developed and validated. Results Four major ellagitannins were quantified in four samples of Lythri herba and in one commercial preparation. The sum of ellagitannins for each sample was determined, which varied from 30.66 to 48.80 mg/g of raw material and 16.57 mg per capsule for the preparation investigated. Conclusion The first validated UHPLC/CAD UHPLC-CAD method for quantification of four major ellagitannins was developed. The universality of the CAD response was evaluated and it is shown that although all compounds analysed have similar structures their CAD response differs significantly. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 30 December 2013 | 2:43 am

Structural Characterisation of Malonyl Flavonols in Leek (Allium porrum L.) Using High-performance Liquid Chromatography and Mass Spectrometry

Introduction Vegetables contain a variety of phytochemicals that have the ability to modify enzymatic and chemical reactions, and therefore may have a positive influence on human health. In particular kaempferol is known to possess anti-carcinogenic activity. Objective The purpose of this work was to determine the structure of glycosylated kaempferol derivatives, acylated with malonic acid on the sugar portion. Methods A methanolic extract of the leaves of Allium porrum L. was submitted to fractionation procedures through semi-preparative HPLC/UV–MS techniques. The collected fractions were evaluated by accurate tandem mass spectrometry experiments using an electrospray ionisation (ESI) quadrupole time-of-flight instrument. Isolated compounds were hydrolysed in order to obtain information on the ester moieties. Results The structures of five compounds not previously reported in leek were determined. The molecules are mono-hexose, di-hexose and coumaroyl, feruloyl and caffeoyl acylated di-hexose derivatives of kaempferol. The common characteristic of the structures relies on the presence of the malonyl moiety on the primary alcoholic function of the sugar immediately linked to the aglycone. Accurate tandem MS experiments and basic hydrolysis treatments revealed a sequence of the acylated glycosidic moieties. Conclusion A set of secondary metabolites of the aerial part of Allium porrum L. (leek) was identified and characterised by ESI/MS2. Knowledge of the presence of these first-reported compounds in leek could provide the means for fully understanding of the metabolism of this plant in relation to the biosynthesis of the phenolics. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 28 December 2013 | 8:47 am

An Overview of Plant Volatile Metabolomics, Sample Treatment and Reporting Considerations with Emphasis on Mechanical Damage and Biological Control of Weeds

Introduction The technology for the collection and analysis of plant-emitted volatiles for understanding chemical cues of plant–plant, plant–insect or plant–microbe interactions has increased over the years. Consequently, the in situ collection, analysis and identification of volatiles are considered integral to elucidation of complex plant communications. Due to the complexity and range of emissions the conditions for consistent emission of volatiles are difficult to standardise. Objective To discuss: evaluation of emitted volatile metabolites as a means of screening potential target- and non-target weeds/plants for insect biological control agents; plant volatile metabolomics to analyse resultant data; importance of considering volatiles from damaged plants; and use of a database for reporting experimental conditions and results. Method Recent literature relating to plant volatiles and plant volatile metabolomics are summarised to provide a basic understanding of how metabolomics can be applied to the study of plant volatiles. Results An overview of plant secondary metabolites, plant volatile metabolomics, analysis of plant volatile metabolomics data and the subsequent input into a database, the roles of plant volatiles, volatile emission as a function of treatment, and the application of plant volatile metabolomics to biological control of invasive weeds. Conclusion It is recommended that in addition to a non-damaged treatment, plants be damaged prior to collecting volatiles to provide the greatest diversity of odours. For the model system provided, optimal volatile emission occurred when the leaf was punctured with a needle. Results stored in a database should include basic environmental conditions or treatments. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 18 December 2013 | 11:30 am

Extraction-free In situ Derivatisation of Timosaponin AIII Using Direct Analysis in Real Time TOF/MS

Introduction Direct analysis in real time (DART) TOF/MS has been used for mass information of various non-polar phytochemicals in raw material with no sample preparation. However, low ionisation efficiency for polar compounds including glycosides limits its extensive use in the field of phytochemical analysis. Objective In order to develop a direct analysis method for polar glycosides using in situ derivatisation, which improves ionisation efficiency of hydrophilic glycosides. Method Anemarrhena Rhizoma was used as a model plant targeting on Timosaponin AIII utilising a Dip-It module. Permethylation was applied to the powdered raw material with tetramethylammonium hydroxide in front of a DART ion source. Also, DART TOF/MS combined with permethylation was applied to timosaponin AIII standard solution to obtain the limit of detection (LOD). Results In situ methylation of timosaponin AIII and Anemarrhena Rhizoma raw material were successfully used to ionise the glycoside. The LOD was found to be in the range of 2.4–4.8 ng for permethylated timosaponin AIII and this level is four times higher than the range of the underivatisation analysis. Direct analysis of permethylated timosaponin from Anemarrhena Rhizoma was also successfully performed. Conclusion A simple and quick derivatisation method with tetramethylammonium hydroxide was developed for the direct identification of a hydrophilic saponin from the plant tissue. Better ionisation efficiency conferred by in situ permethylation enabled ionisation of whole molecules of timosaponin AIII from the plant tissue. This simple analytical method will provide a solution to reduce tedious sample preparation steps, not only for non-polar but also hydrophilic natural products directly from the tissue. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 5 December 2013 | 9:26 am

Stereochemistry of C18 Monounsaturated Cork Suberin Acids Determined by Spectroscopic Techniques Including 1H-NMR Multiplet Analysis of Olefinic Protons

Introduction Suberin is a biopolyester responsible for the protection of secondary plant tissues, and yet its molecular structure remains unknown. The C18:1 ?-hydroxyacid and the C18:1 ?,?-diacid are major monomers in the suberin structure, but the configuration of the double bond remains to be elucidated. Objective To unequivocally define the configuration of the C18:1 suberin acids. Methods Pure C18:1 ?-hydroxyacid and C18:1 ?,?-diacid, isolated from cork suberin, and two structurally very close C18:1 model compounds of known stereochemistry, methyl oleate and methyl elaidate, were analysed by NMR spectroscopy, Fourier transform infrared (FTIR) and Raman spectroscopy, and GC–MS. Results The GC–MS analysis showed that both acids were present in cork suberin as only one geometric isomer. The analysis of dimethyloxazoline (DMOX) and picolinyl derivatives proved the double bond position to be at C–9. The FTIR spectra were concordant with a cis-configuration for both suberin acids, but their unambiguous stereochemical assignment came from the NMR analysis: (i) the chemical shifts of the allylic 13C carbons were shielded comparatively to the trans model compound, and (ii) the complex multiplets of the olefinic protons could be simulated only with 3JHH and long-range 4JHH coupling constants typical of a cis geometry. Conclusion The two C18:1 suberin acids in cork are (Z)-18-hydroxyoctadec-9-enoic acid and (Z)-octadec-9-enedoic acid. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 5 December 2013 | 9:26 am

Determination of Salicinoids by Micro-high-performance Liquid Chromatography and Photodiode Array Detection

Introduction Plant-derived salicinoids (conjugates of glucose and salicylate phenolic moieties) are potent modulators of plant–herbivore interactions. We demonstrate the use of micro-high-performance liquid chromatography (?HPLC) with photodiode-array detection (DAD) for quantification of four salicinoids (salicin, salicortin, hydroxycyclohexen-on-oyl salicortin and tremulacin) in methanolic extracts of Populus. Objective To develop and implement a solvent-conserving ?HPLC method to quantify salicinoids in methanolic extracts of Populus tissue. Methods Salicinoids were extracted from Populus tissue into methanol, filtered, and introduced to ?HPLC. Extracted analytes were separated on a Zorbax SB C18-column with a binary gradient of methanol and water (with 2% tetrahydrofuran; 20 ?L/min), and quantified by DAD (274 nm). We confirmed measurement reliability through standard addition, comparison with an accepted method, and assessment of chromatographic peak purity by ultraviolet absorbance spectra. Results Method detection and quantification limits for the salicinoids as a percentage of dry leaf weight were as follows: salicin (0.1%, 0.2%), salicortin (0.001%, 0.02%), hydroxycyclohexen-on-oyl salicortin (0.02%, 0.06%) and tremulacin (0.0006%, 0.002%). Calibrations by external standardisation were linear over 1.5 orders of magnitude with acceptable accuracy and reproducibility. Conclusion Micro-HPLC can serve as a solvent-conserving alternative to conventional HPLC for quantification of salicinoids in Populus tissue. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 26 November 2013 | 6:41 am

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