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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:

Recognition of Pyrrolizidine Alkaloid Esters in the Invasive Aquatic Plant Gymnocoronis spilanthoides (Asteraceae)

Introduction The freshwater aquatic plant Gymnocoronis spilanthoides (Senegal tea plant, jazmín del bańado, Falscher Wasserfreund) is an invasive plant in many countries. Behavioural observations of pyrrolizidine alkaloid-pharmacophagous butterflies suggested the presence of pyrrolizidine alkaloids in the plant. Objective To determine whether the attraction of the butterflies to the plant is an accurate indicator of pyrrolizidine alkaloids in G. spilanthoides. Methods The alkaloid fraction of a methanolic extract of G. spilanthoides was analysed using HPLC with electrospray ionisation MS and MS/MS. Two HPLC approaches were used, that is, a C18 reversed-phase column with an acidic mobile phase, and a porous graphitic carbon column with a basic mobile phase. Results Pyrrolizidine alkaloids were confirmed, with the free base forms more prevalent than the N-oxides. The major alkaloids detected were lycopsamine and intermedine. The porous graphitic carbon HPLC column, with basic mobile phase conditions, resulted in better resolution of more pyrrolizidine alkaloids including rinderine, the heliotridine-based epimer of intermedine. Based on the MS/MS and high-resolution MS data, gymnocoronine was tentatively identified as an unusual C9 retronecine ester with 2,3-dihydroxy-2-propenylbutanoic acid. Among several minor-abundance monoester pyrrolizidines recognised, spilanthine was tentatively identified as an ester of isoretronecanol with the unusual 2-acetoxymethylbutanoic acid. Conclusions The butterflies proved to be reliable indicators for the presence of pro-toxic 1,2-dehydropyrrolizidine alkaloids in G. spilanthoides, the first aquatic plant shown to produce these alkaloids. The presence of the anti-herbivory alkaloids may contribute to the plant's invasive capabilities and would certainly be a consideration in any risk assessment of deliberate utilisation of the plant. The prolific growth of the plant and the structural diversity of its pyrrolizidine alkaloids may make it ideal for investigating biosynthetic pathways or for large-scale production of specific alkaloids. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 27 January 2015 | 9:47 am

Systematic Separation and Purification of Iridoid Glycosides and Crocetin Derivatives from Gardenia jasminoides Ellis by High-speed Counter-current Chromatography

Introduction Iridoid glycosides and crocetin derivatives are the main bioactive components of Gardenia. The processes of separation of these compounds reported in much of the literature are tedious, time consuming and require multiple chromatographic steps, which results in lower recovery and higher costs. Objective To develop a high-speed counter-current chromatography (HSCCC) method for the systematic separation and purification of iridoid glycosides and crocetin derivatives on a preparative scale from Gardenia. Methods After fractionation using HPD100 column chromatography, n-butanol:ethanol:water (10:1:10, v/v) was selected to purify gardenoside, 6?-hydroxy geniposide and geniposidic acid from fraction A; ethyl acetate:n-butanol:water (2:1.5:3, v/v) was used to isolate geniposide from fraction B; crocin-1, crocin-2, crocin-3 and crocin-4 were purified by hexane:ethyl acetate:n-butanol:water (1:2:1:5, v/v) from fraction C. The head-to-tail elution mode was used with a flow rate of 8.0?mL/min and a rotary speed of 600?rpm. Results After HSCCC isolation, 151.1?mg of gardenoside, 52.2?mg of 6?-hydroxy geniposide and 24.5?mg of geniposidic acid were obtained from 800?mg of fraction A; 587.2?mg of geniposide was obtained from 800?mg of Fraction B; 246.2?mg of crocin-1, 34.2?mg of crocin-2, 24.4?mg of crocin-3 and 24.7?mg of crocin-4 were obtained from 1000?mg of fraction C. Their purities were found by UPLC analysis to be 91.7%, 93.4%, 92.5%, 98.2%, 94.1%, 96.3%, 94.1% and 98.9% respectively. Conclusion The present results demonstrates that the main iridoid glycosides and crocetin derivatives in Gardenia can be obtained efficiently from extracts using HSCCC. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 27 January 2015 | 9:19 am

Non-targeted Molecular Characterisation of a Rose Flower Ethyl Acetate Extract Using Ultra-HPLC with Atmospheric Pressure Photoionisation and Quadrupole Time-of-flight MS/MS

Introduction A non-targeted approach to characterise the phytochemical composition of the flower organ of an original rose cultivar ‘Jardin de Granville’® was developed. Particular attention was paid to the less documented molecular families of intermediate polarity, compared with the polyphenol family (anthocyanins, flavonoids, tannins) and volatile compounds. Objective To develop a molecular fingerprinting method for the rapid qualitative phytochemical characterisation of the rose flower ethyl acetate extract. Material and methods An ultra-HPLC with atmospheric pressure photoionisation (APPI) and quadrupole time-of-flight (QTOF) MS/MS combined with microwave-assisted extraction was carried out for ethyl acetate extracts as an intermediate polarity extraction solvent in order to obtain the most exhaustive extract containing a large range of molecular families. An optimised methodology based on the coupling of the UHPLC and APPI source with a QTOF analyser was developed to characterise the extracted molecules. Results Sixty-one compounds were identified in the extract, covering eight molecular families of intermediate polarity ranging from polyphenols to triglycerides. The presence of flavonoids with anti-oxidant properties and of triterpenoids with potential anti-inflammatory activity was evidenced and cell-wall constituents such as fatty acids, glycolipids, sphingolipids and acylated sterol glycosides were characterised. Some chlorophyll derivatives were also detected. Conclusion The method developed is appropriate for fast phytochemical evaluation of rose ethyl acetate extract. It produced accurate mass and MS/MS spectra, which permitted identification of a wide range of compounds of intermediate polarity. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 27 January 2015 | 8:43 am

LC–MS/MS Quantitative Determination of Tetrapterys mucronata Alkaloids, a Plant Occasionally used in Ayahuasca Preparation

Introduction Tetrapterys mucronata Cav. (Malpighiaceae) is a plant used in some regions of Brazil in the preparation of ayahuasca. Objective To determine the content of the main tryptamine alkaloids in the stem bark of T. mucronata Cav. and assess their possible toxic and hallucinogenic properties based on the doses found in a water decoction that mimics the ayahuasca preparation. Methods Four alkaloids previously described for their toxic and hallucinogenic properties were quantitated by multiple reaction monitoring HPLC combined with electrospray ionisation and tandem MS (HPLC–ESI/MS/MS) in the water decoction and ethanolic extracts from the bark of T. mucronata. Results Exhaustive extraction of the stem barks with ethanol revealed the following alkaloid levels: bufotenine (1) 3.26?±?0.31?mg/g, 5-methoxy-N-methyltryptamine (2) 0.88?±?0.08?mg/g, 5-methoxy-bufotenine (3) 3.07?±?0.22?mg/g and 2-methyl-6-methoxy-1,2,3,4-tetrahydro-?-carboline (4) 0.14?±?0.004?mg/g. The water decoction presented slightly lower levels, ranging between 2.32?±?0.14, 0.50?±?0.04, 1.53?±?0.09 and 0.10?±?0.01?mg/g for (1), (2), (3) and (4) respectively. Conclusions The HPLC–ESI/MS/MS quantitation revealed significant alkaloid levels, in particular for bufotenine and 5-methoxy-bufotenine. As such compounds are known for their toxic and hallucinogenic properties, these results indicate that the consumption of this plant as an ingredient in ayahuasca preparations may present a risk to consumers. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 26 January 2015 | 6:22 am

Trace Determination of Lead, Chromium and Cadmium in Herbal Medicines Using Ultrasound-Assisted Emulsification Microextraction Combined with Graphite Furnace Atomic Absorption Spectrometry

Introduction The World Health Organization (WHO) recommends that medicinal plants should be checked for the presence of heavy metals. A preconcentration and separation technique for trace amounts of heavy metals from plant matrix is necessary in order to increase the sensitivity and precision of their determination. Objective Lead, chromium and cadmium contaminations in herbal medicines were monitored using ultrasound-assisted emulsification microextraction (USAEME) combined with graphite furnace atomic absorption spectrometry (GF–AAS). Methods In this work, the metal ions in the aqueous solution were complexed with ammonium pyrrolidine dithiocarbamate (APDC) and were extracted into 45??L of toluene that was sonically dispersed in the aqueous phase. The emulsion formed was centrifuged and 20??L of separated toluene was injected into a GF–AAS for analysis. Several factors including the kind of extraction solvent and its volume, sample pH, ionic strength and concentration of APDC were optimised. Results The linear dynamic range (LDR) values were in the range of 0.05 to 20?µg/L and the limit of detection values were in the range of 0.002–0.03?µg/L for target heavy metals. Enrichment factors were obtained in the range of 70–500. The precision of the proposed method was ?8% (n?=?5). The obtained amounts of Pb, Cr and Cd in selected herbal medicines were in the standard range, according to the WHO reports. Conclusion The USAEME with GF–AAS procedure was shown to be an efficient, rapid, inexpensive and eco-friendly method for the determination of lead, chromium and cadmium in herbal medicines. Application of the USAEME method leads to an increased extraction efficiency with satisfactory precision in a short time using an extraction solvent volume at the microlitre level. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 9 January 2015 | 12:23 pm

Qualitative and Quantitative Analysis of Potentilla fulgens Roots by NMR, Matrix-assisted Laser Desorption/Ionisation with Time-of-Flight MS, Electrospray Ionisation MS/MS and HPLC/UV

Introduction Potentilla fulgens is a commonly used folk medicine by natives of northeast India, Nepal and Bhutan and is rich in polyphenolic and triterpene constituents. Objective To identify chemomarkers in the roots of P. fulgens by an interplay of 13C-NMR, matrix-assisted laser desorption/ionisation with time-of-flight (MALDI/TOF) MS, electrospray ionisation (ESI) MS/MS and HPLC/UV. Material and methods The 13C-NMR spectrum of crude methanolic extract was recorded in deuterated dimethyl sulphoxide. For MALDI/TOF/MS analysis, 2,5-dihydroxybenzoic acid was used as the matrix. For determination of chemical constituents, two independent simple isocratic HPLC/UV methods for monomeric/oligomeric flavanols and triterpene acids were developed and validated. Results The 13C-NMR spectrum of the methanolic extract indicated the presence of B-type oligomeric polyphenolics containing mainly epicatechin/catechin (epicat/cat) and epiafzelechin/afzelechin (epiafz/afz) as the monomeric units. Several isobaric monomeric and oligomeric flavanols and triterpenoids were tentatively identified by MALDI/TOF/MS and ESI/MS/MS. Fourteen compounds (four monomeric and five dimeric flavanols and five triterpene acids) were isolated using repeated column chromatography and semi-preparative HPLC, and were quantitated using HPLC/UV. Conclusion It is evident from these analyses that roots of P. fulgens contain flavans, including oligomeric flavanols, as major constituents followed by triterpene acids. The methods described can be applied to other Potentilla species to identify their constituents. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 16 December 2014 | 11:57 am

Non-targeted Metabolomic Profile of Fagus Sylvatica L. Leaves using Liquid Chromatography with Mass Spectrometry and Gas Chromatography with Mass Spectrometry

Introduction Fagus sylvatica L. is one of the most widely distributed broad-leaved tree species in central and western Europe, important to the forest sector and an accurate biomarker of climate change. Objective To profile the beech leaf metabolome for future studies in order to investigate deeper into the characterisation of its metabolic response. Methods Leaf extracts were analysed using LC–MS by electrospray ionisation in negative mode from m/z 100–1700 and GC–MS by electron ionisation in scan mode from m/z 35–800. Results The LC–MS profile resulted in 56 compounds, of which 43 were identified and/or structurally characterised, including hydroxycinnamic acid derivatives, flavan-3-ols and proanthocyanidins, and flavonols. From a second analysis based on GC–MS, a total of 111 compounds were identified, including carbohydrates, polyalcohols, amino acids, organic acids, fatty acids, phenolic compounds, terpenoids, sterols and other related compounds. Many of the compounds identified were primary metabolites involved in major plant metabolic pathways, however, some secondary metabolites were also detected. Some of them play roles as tolerance-response osmoregulators and osmoprotectors in abiotic stress, or as anti-oxidants that reduce the effect of reactive oxygen species and promote many protective functions in plants. Conclusions This study provides a broad and relevant insight into the metabolic status of F. sylvatica leaves, and serves as a base for future studies on physiological and molecular mechanisms involved in biotic or abiotic stress. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 16 December 2014 | 11:57 am

Isolation of Flavonoids and Triterpenoids from the Fruits of Alphitonia Neocaledonica and Evaluation of their Anti-oxidant, Anti-tyrosinase and Cytotoxic Activities

Introduction Alphitonia neocaledonica (Rhamnaceae) is an endemic tree of New Caledonia. Although three flavonoids have been identified in the leaves, the secondary metabolite profile of the fruits has never been investigated. Objective Phytochemical investigation of A. neocaledonica fruits and evaluation of their anti-oxidant, anti-tyrosinase and cytotoxic activities. Methods A hydromethanolic extract was fractionated by liquid–liquid extraction to obtain ethyl acetate and n-butanolic fractions. The ethyl-acetate-soluble part was purified by silica-gel column chromatography and high-performance liquid chromatography (HPLC). The n-butanol-soluble part was fractionated by centrifugal partition extraction (CPE) and the collected fractions were further purified by centrifugal partition chromatography (CPC) and HPLC. The chemical structures of the purified compounds were identified by nuclear magnetic resonance and mass spectrometry. Results Three triterpenoids and one flavonoid were isolated from the ethyl-acetate-soluble part. Fractions enriched in triterpenoids, flavonoids and catechin derivatives were obtained from the n-butanol-soluble part. Gallocatechin and flavonoids were obtained as pure compounds by further CPC and HPLC purification. The n-butanolic-soluble part showed anti-oxidant and anti-tyrosinase activities due to the presence of tannins and gallocatechin. The triterpenoid alphitolic acid showed a moderate cytotoxic activity against KB cell line (median inhibition concentration?=?8.5??M). Conclusions Nine known compounds including three triterpenes, five flavonoids and (+) gallocatechin, as well as a new 3-O-(6-E-feruloyl)-?-D-glucopyranosyl-(1??2)-[?-D-xylopyranosyl-(1??2)-]?-L-rhamnopyranosyl-quercetin, were isolated from A. neocaledonia fruits. The hydromethanolic extract possesses a potential cytotoxic activity due to the presence of triterpenes, and it can also be valuable as a cosmetic ingredient for its anti-oxidant and anti-tyrosinase activities. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 16 December 2014 | 11:57 am

Ultrasound-assisted Extraction of Gymnemic Acids from Gymnema sylvestre Leaves and its Effect on Insulin-producing RINm-5?F ? Cell Lines

Introduction Gymnema sylvestre is an important anti-diabetic medicinal plant, hence it is necessary to study the effective extraction of its active medicinal components. Objective To develop an efficient ultrasound-assisted extraction method for anti-diabetic gymnemic acids from Gymnema sylvestre leaves and measure their effect on insulin-producing RINm-5?F ? cells. Methods Box–Behnken's design and response surface methodology was applied to the ultrasound-assisted extraction of gymnemic acids from Gymnema sylvestre leaves. Analysis of gymnemic acids was carried out by high-performance thin-layer chromatography by converting total gymnemic acids into gymnemagenin by alkali hydrolysis. Effects of extracts on insulin production were tested on cultured, insulin-producing RINm-5?F ? cell lines. Results The point prediction tool of the design expert software predicted 397.9?mg gymnemic acids per gram of the defatted G. sylvestre leaves using ultrasound-assisted extraction, with ethanol at 60?°C for 30?min. The predicted condition shows 93.34% validity under experimental conditions. The ultrasound-assisted extract caused up to about four times more insulin production from RINm-5?F ? cells than extracts obtained from Soxhlet extraction. Conclusions Response surface methodology was successfully used to improve the extraction of gymnemic acids from G. sylvestre leaves. The ultrasound-assisted extraction process may be a better alternative to prepare such herbal extracts because it saves time and may prevent excess degradation of the target analytes. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 3 December 2014 | 11:23 am

Chemical Differentiation and Quality Evaluation of Commercial Asian and American Ginsengs based on a UHPLC–QTOF/MS/MS Metabolomics Approach

Introduction Asian and American ginsengs are widely used medicinal materials and are being used more and more in health products. The two materials look alike but function differently. Various forms of both types of ginseng are found in the market, causing confusion for consumers in their choice. Objective To evaluate the overall quality of commercial Asian and American ginsengs and investigate the characteristic chemical markers for differentiating between them. Methods This article investigated 17 Asian and 21 American ginseng samples using an ultra-HPLC combined with quadrupole time-of-flight MS/MS technique. The data were processed by principal component analysis and orthogonal partial least squared discriminant analysis. Results In the chromatograms, a total of 40 peaks were detected. Among them, six were positively identified, and all of the remainder were tentatively identified. According to statistical results, ginsenosides Rf, Rb2 and Rc together with their isomers and derivatives were more likely to be present in Asian ginsengs, whereas ginsenoside Rb1, pseudoginsenoside F11 and ginsenoside Rd together with their isomers and derivatives tended to be present in American ginsengs. For Asian ginsengs, ginsenoside Ra3 and 20-?-D-glucopyranosyl-ginsenoside-Rf were more likely to be present in forest samples, whereas contents of floralquinquenoside B, ginsenosides Ro and Rc, and zingibroside R1 were higher in sun-dried ginsengs. For American ginseng, wild samples often had more of the notoginsenosides R1 and Rw2 and less of the ginsenosides Rd, Rd isomer and 20 (S)-Rg3 than cultivated samples. Conclusion The method provided important fingerprint information for authentication and evaluation of Asian and American ginsengs from various commercial products. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 2 December 2014 | 7:02 am

The Evaluation of Reduction of Fe(III) in 3-Hydroxy-4-Nitroso-2,7-Naphthalene Disulphonic Medium as an Alternative Ferric Reducing Activity Power Assay

Introduction The growing interest in determination of anti-oxidant capacity through non-labour, effective and less costly methods encouraged the development of the spectrophotometric procedure presented in this study. Objective To investigate the reduction reaction of Fe(III) in 3-hydroxy-4-nitroso-2,7-naphthalenedisulphonic anion (NRS) medium as an alternative ferric reducing activity power (FRAP) assay for determining the total reduction capacity (RC). Materials and methods The absorbance values at 730?nm were used to determine the RC of aqueous extracts of nine Brazilian plants. The results were compared with the values obtained with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and with the total polyphenol content (TPC). The RCs of phenolic derivatives, flavonoids, amino acids and other anti-oxidant compounds were determined. Results Paired t-test with RC values obtained with both assays (proposed FRAP and DPPH) showed no statistically significant difference. In addition, the RC values from the proposed FRAP assay are proportional to those found with TPC values (r =0.916). In addition, the conditional reduction potential of the Fe(III)/Fe(II) couple (0.685?V vs NHE (normal hydrogen electrode)) and the molar absorptivities at 730?nm of the Fe(NRS)33? and Fe(NRS)34? complexes (1.88?×?103 and 1.77?×?104?L/cm?×?mol, respectively) were calculated because these values were not available. Conclusion The proposed assay is adequate for determination of the RC of plant extracts, and the results infer that other samples derived from plants (e.g. beers and wines) and even biological samples (e.g. serum and urine) also could be analysed. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 27 November 2014 | 9:07 am

Development and Validation of Liquid Chromatography Combined with Tandem Mass Spectrometry Methods for the Quantitation of Simalikalactone E in Extracts of Quassia amara L. and in Mouse Blood

Introduction Simalikalactone E (SkE) from Quassia amara, has been proved to be a valuable anti-malarial and anti-cancer compound. As SkE is very scarce, methods of quantitation are needed in order to optimise its isolation process and to determine pharmacokinetic data. Objective To validate methods using liquid chromatography coupled to mass spectrometry for the quantitation of SkE in plant extracts and in biological fluids. Methods High- and ultrahigh-performance liquid chromatography (UHPLC) coupled to ion trap mass spectrometry (MS) with single ion monitoring detection and to triple quadrupole-linear ion trap tandem mass spectrometry with multiple reaction monitoring detection methods were developed. Validation procedure was realised according to the International Conference on Harmonisation guideline. Methanol extracts of dried Quassia amara leaves, and mouse-blood samples obtained after various routes of administration, were analysed for SkE. Results Methods were validated and gave similar results regarding the content of SkE expressed per kilogram of dry leaves in the traditional decoction (160?±?12?mg/kg) and in the methanol extract (93?±?2?mg/kg). The recovery of the analyte from mouse blood ranged from 80.7 to 119.8%. Simalikalactone E was only detected using UHPLC–MS/MS (0.2?±?0.03?mg/L) in mouse blood after intravenous injection: none was detected following intraperitoneal or oral gavage administration of SkE. Conclusion The LC–MS methods were used for the quantitation of SkE in plant extracts and in mouse blood. These methods open the way for further protocol optimisation of SkE extraction and the determination of its pharmacokinetic data. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 27 November 2014 | 9:06 am

Simultaneous UHPLC/DAD/(+/?)HESI–MS/MS Analysis of Phenolic Acids and Nepetalactones in Methanol Extracts of Nepeta Species: A Possible Application in Chemotaxonomic Studies

Introduction Nepeta species contain a variety of secondary metabolites, including iridoid monoterpenes – nepetalactones and phenolic acids – that are considered the main bioactive constituents. This work represents the first attempt to comparatively explore variations in these two major groups of secondary metabolites within the genus. Objective To develop an efficient analytical methodology for simultaneous analysis of nepetalactones and phenolic acids in methanol extracts of selected Nepeta species, and to evaluate its potential application in chemotaxonomic studies. Material and methods A UHPLC combined with linear-trap quadrupole (LTQ) orbitrap MS method was used to characterise chemical diversity and complexity of phenolics among 12 selected Nepeta species. A targeted metabolomic approach using UHPLC coupled to a diode array detector (DAD) and combined with (+/?) heated electrospray ionisation (HESI) MS/MS was developed and validated for quantitative analysis of six hydroxycinnamic acid derivatives and four nepetalactones. Results Phenolic profiling provided a valuable database of bioactive compounds in the plant group studied, including phenolic acids (hydroxybenzoic and hydroxycinnamic acids) and flavonoids (flavones, flavonols and flavanones). Principal component analysis and cluster analysis suggested the applicability of 10 targeted compounds as chemomarkers for chemotaxonomic studies. Pearson's correlation analysis revealed significant positive correlations between metabolites involved in different biosynthetic pathways (phenylpropanoid or monoterpenoid). Conclusion The described targeted metabolomic approach proved to be highly beneficial in designing a phytochemical overview of the genus Nepeta, and might have applications in further clarification of phylogenetic relations. Furthermore, it has the potential to be implemented in a routine quality control of plant material and herbal preparations. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 27 November 2014 | 8:14 am

In Situ Detection and Identification of Hesperidin Crystals in Satsuma Mandarin (Citrus unshiu) Peel Cells

Introduction Hesperidin, a flavonoid known to have important pharmacological effects, accumulates particularly in the peels of satsuma mandarin (Citrus unshiu). Although histochemical studies have suggested that hesperidin forms crystals in some tissues of the Rutaceae and Umbelliferae, there has been no rigorous in situ detection or identification of hesperidin crystals in C. unshiu. Objective To characterise the chemical component of the crystals found in C. unshiu peels using Raman microscopy. Methods Sections of C. unshiu peels were made. The distribution and morphology of crystals in the sections were analysed microscopically. Raman microscopy was used to detect hesperidin in the sections directly. Results The crystals were more abundant in immature peel and were observed particularly in areas surrounding vascular bundles, around the border between the flavedo and albedo layers and just below the epidermal cells. In the morphological analysis by scanning electron microscopy, needle-shaped crystals aggregated and formed clusters of spherical crystals. Spectra obtained by Raman microscopy of the crystals in the peel sections were consistent with those of the hesperidin standard. Conclusion This study showed the detailed distribution of crystals in C. unshiu peels and their main component was identified using Raman microscopy to be hesperidin for the first time. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 6 November 2014 | 10:18 am

Ultrahigh-performance Liquid Chromatography and Mass Spectrometry (UHPLC–LTQ/Orbitrap/MS/MS) Study of Phenolic Profile of Serbian Poplar Type Propolis

Introduction Propolis is a resinous natural substance collected by honeybees from different plant sources. Due to the presence of various phytochemicals, this bee-product exhibits numerous biological activities, including anti-bacterial, anti-viral, anti-inflammatory, anti-oxidant, immunostimulating and anti-tumour effects. As the chemical composition and biological activity of propolis depend on its botanical and geographical origin, searching for new bioactive substances in various types of propolis from unexplored regions is of great importance. Objective The aim of this study is the evaluation of the phenolic profile of poplar propolis samples in order to characterise Serbian propolis, to identify possible new constituents and to specify the phenolic components relevant for differentiation of poplar propolis samples into two subgroups through simultaneous analysis of poplar bud extracts. Methods Ethanolic extracts of propolis and poplar buds were comprehensively analysed using ultrahigh-performance liquid chromatography coupled with hybrid mass spectrometry, which combines the linear trap quadrupole and Orbitrap MS/MS mass analyser together with chemometric methods. Results Extensive fingerprint analysis of Serbian propolis was achieved for the first time. Seventy-five phenolic compounds were detected. Eight of them were identified in propolis for the first time. Pattern-recognition methods applied to the content of ten quantified phenolics verified the existence of two subgroups of propolis, with galangin, chrysin and pinocembrin as the most influential distinguishing factors. Conclusion The phenolic composition of the analysed propolis samples confirm their affiliation to the European poplar type propolis and the existence of two subgroups according to botanical origin. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 6 November 2014 | 10:17 am

Revealing Metabolomic Variations in Cortex Moutan from Different Root Parts using HPLC–MS method

Introduction The distribution of metabolites in the different root parts of Cortex Moutan (the root bark of Paeonia suffruticosa Andrews) is not well understood, therefore, scientific evidence is not available for quality assessment of Cortex Moutan. Objective To reveal metabolomic variations in Cortex Moutan in order to gain deeper insights to enable quality control. Methods Metabolomic variations in the different root parts of Cortex Moutan were characterised using high-performance liquid chromatography combined with mass spectrometry (HPLC–MS) and multivariate data analysis. The discriminating metabolites in different root parts were evaluated by the one-way analysis of variance and a fold change parameter. Results The metabolite profiles of Cortex Moutan were largely dominated by five primary and 41 secondary metabolites . Higher levels of malic acid, gallic acid and mudanoside-B were mainly observed in the second lateral roots, whereas dihydroxyacetophenone, benzoyloxypaeoniflorin, suffruticoside-A, kaempferol dihexoside, mudanpioside E and mudanpioside J accumulated in the first lateral and axial roots. The highest contents of paeonol, galloyloxypaeoniflorin and procyanidin B were detected in the axial roots. Accordingly, metabolite compositions of Cortex Moutan were found to vary among different root parts. Conclusion The axial roots have higher quality than the lateral roots in Cortex Moutan due to the accumulation of bioactive secondary metabolites associated with plant physiology. These findings provided important scientific evidence for grading Cortex Moutan on the general market. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 17 September 2014 | 1:41 pm

Comparison of Soxhlet, Accelerated Solvent and Supercritical Fluid Extraction Techniques for Volatile (GC–MS and GC/FID) and Phenolic Compounds (HPLC–ESI/MS/MS) from Lamiaceae Species

Introduction Plants from the Lamiaceae family have been known traditionally for their beneficial health-promoting properties, attributed to their anti-inflammatory, anaesthetic and anti-microbial effects. Objective The purposes of this study was to characterise the essential oils from four Lamiaceae plants by applying different extraction techniques. Methods Accelerated solvent (ASE), Soxhlet and supercritical fluid (SFE) extraction methods were compared for their efficiency in obtaining the essential oils from plants. The volatile compounds were identified by GC–MS and the main chemotype was quantified by GC with flame ionisation detection (FID). Phenolic compounds were identified and quantified by HPLC and electrospray ionisation (ESI) with MS/MS. Results The essential oils Mentha piperita (ct. menthol/menthone), Rosmarinus officinalis L. (ct. eucalyptol/camphor) and Origanum vulgare (ct. carvacrol/thymol), whereas Thymus vulgaris L. was found to be a pure chemotype (ct. thymol). All three extracts also contained six phenolic compounds. The highest extraction yields were achieved by the Soxhlet and ASE techniques, with M. piperita and R. officinalis L. producing the highest concentrations of rosmarinic and carnosic acids. Finally, it was observed that M. piperita and O. vulgare produced the highest total phenolic content, whereas R. officinalis L. and T. vulgaris L. produced the highest anti-oxidant activity. Conclusion The ASE and Soxhlet extraction techniques presented the highest yields of volatile and phenolic compounds, showing their suitability to characterise the chemical profile of aromatic plants. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 4 September 2014 | 4:07 pm

Enantiomeric Determination of Four Diastereoisomeric Oxyneolignans from Bambusa tuldoides Munro

Introduction Bambusa tuldoides Munro, a bamboo species, is used as a health food, dietary supplement and folk medicine in China, and produces lignans that can be used to supplement other natural sources. Objective To simultaneously separate eight stereoisomers of a particular type of oxyneolignan by chiral chromatography. Methods Ninety-five per cent ethanol extracts of B. tuldoides Munro were analysed using HPLC/UV with a chiral column. The structures and configurations of isolated compounds were elucidated using NMR and circular dichroism (CD). Results Four diastereoisomers were characterised and given the names oxyneolignans A, B, C and D. Furthermore, each oxyneolignan occurred as a pair of enantiomers. The oxyneolignans A–D consisted of the erythro-diastereoisomer of oxyneolignan at C7 and C8. Conclusion The chiral chromatography combined with the analysis techniques of NMR and CD reported here were reliable methods for discovering and separating the enantiomers. Copyright © 2014 John Wiley & Sons, Ltd.

Posted on 4 September 2014 | 1:56 pm

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

Introduction Myrosinase (thioglucoside glucohydrolase; E.C., 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

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

Introduction Quantitative 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. Objective To establish a qNMR method for purity tests of six dammarane-type ginsenoside standards. Methods Several 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. Conclusion The 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

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

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