Phytochemical Analysis - current research articles

Identification and Characterisation of Phenolics in Polygonum capitatum by Ultrahigh-Performance Liquid Chromatography with Photodiode Array Detection and Tandem Mass Spectrometry

Introduction Polygonum capitatum is a well-known Chinese medicinal plant widely used by the Miao people for the treatment of various urologic disorders. Previous investigations have shown the presence of various types of phenolics. Our ultrahigh-performance liquid chromatography with photodiode array detection and mass spectrometry (UPLC–PDA–MS) analysis indicated that flavonoid glycosides and polyphenolic glycosides were its major constituents and quite a number of phenolic compounds have not yet been identified. Identification or characterisation of the major compounds of this plant will contribute to the scientific understanding of the medicinal plant and the authentication of the plant material and its pharmaceutical preparations. Objective To develop an efficient method for the identification and structural characterisation of the major compounds present in P. capitatum. Methods Elution of the 70% ethanol extract of P. capitatum by 80% ethanol on a D101 macroporous resin column afforded a phenolics-enriched fraction, separation of which by Sephadex LH-20 column chromatography eluted with absolute ethanol gave a tannin-free phenolic fraction (TFPF). Compounds present in TFPF were identified and structurally characterised by UPLC–PDA–ESI–MS/MS. Results An amino acid and 40 phenolics including a number of flavonoid glycosides and polyphenolic glycosides were identified or structurally characterised in TFPF. Among these compounds, four were new and 19 were described in the plant for the first time. Conclusion The study showed that TFPF of P. capitatum contained flavonoid glycosides and polyphenolic glycosides as its major principles. Polyphenolic glycosides were perhaps the most typical chemical markers of P. capitatum. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 23 May 2013 | 1:17 pm

1H-NMR Quantification of Major Saccharides in Açaí Raw Materials: a Comparison of the Internal Standard Methodology with the Absolute Intensity qNMR Method

Introduction While the use of internal standard methodology for qNMR is a proven and reliable form of quantification, simplified alternative approaches are needed. Agilent's absolute intensity qNMR utility software is a valuable alternative that has not yet been subjected to validation in the peer-reviewed literature. Objective To provide validation of Agilent's absolute intensity qNMR method with a specific application to natural product quantification by measuring saccharide content in açaí materials. Methods In order to validate the method, calibration test samples of ibuprofen were prepared in DMSO-d6 at nine different concentrations and measured with 1H-NMR. A minimum of 40 spectra were collected for each sample, and the absolute intensity utility was used for quantification. The same methodology was then applied to the açaí materials, creating triplicates for each of the materials and using 3-(trimethylsilyl)-1-propanesulphonic acid sodium salt in water-d2 as both the solvent and internal standard. 1H-NMR spectra were collected, and the amounts of glucose, sucrose and fructose were determined using both the internal standard approach and the absolute intensity qNMR method. Results Applying the absolute intensity utility to the ibuprofen samples demonstrated a linear response (R2?=?0.99943). For the açaí investigations, results obtained from the absolute intensity method were comparable to those obtained from the internal standard approach, with percentage differences ranging from 0.5–6.2%. Conclusion This study demonstrates the accuracy, precision and reliability of Agilent's absolute intensity qNMR method. In addition, practical information is provided for assessing the saccharide contents of açaí materials. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 23 May 2013 | 12:50 pm

1H-NMR Fingerprinting of Vaccinium vitis-idaea Flavonol Glycosides

Introduction The fruits of Vaccinium vitis-idaea L. are a valuable source of biologically active flavonoid derivatives. For studies focused on the purification of its quercetin glycosides (QGs) and related glycosides from plants and for the purpose of biological studies, the availability of numeric datasets from computer-assisted 1H iterative full spin analysis (HiFSA), that is, 1H-NMR fingerprinting, can replace and assist the repetitive and tedious two-dimensional NMR identification protocol required for both known and new compounds, respectively. Objective To fully interpret the complex 1H-NMR fingerprints of eight QGs obtained from the berries of V. vitis-idaea and provide complete and unambiguous signal assignments. Methods Vaccinium vitis-idaea QGs were purified in a single run by long-bed gel permeation chromatography and identified by comparison with commercially available compounds using LC–MS combining ion-trap and time-of-flight detection and one- or two-dimensional NMR. The HiFSA analysis yielded full sets of 1H chemical shifts and proton–proton coupling constants, allowing for field-independent spectral simulation. Results Signal assignments were achieved for the reference standards and the QGs that dominated in purified fractions. However, even mixtures of two to three QGs could be fitted using the HiFSA approach. In the case of the overlapped sugar resonances, the initial fitting of the 1H spectra of reference compounds, together with values extracted from the two-dimensional NMR data and literature data, assisted in the process. Conclusion The HiFSA method revealed for the first time the presence of Q-3-O-?-glucopyranoside and Q-3-O-?-glucuronopyranoside in the berries of V. vitis-idaea, and unambiguously confirmed the structures of Q-3-O-[4?-(3-hydroxy-3-methylglutaroyl)]-?-rhamnopyranoside, Q-3-O-?-rhamnopyranoside, Q-3-O-?-galactopyranoside, Q-3-O-?-arabinofuranoside, Q-3-O-?-xylopyranoside and Q-3-O-?-arabinopyranoside. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 23 May 2013 | 12:43 pm

A Proposed Biosynthetic Pathway of Picrosides Linked through the Detection of Biochemical Intermediates in the Endangered Medicinal Herb Picrorhiza kurroa

Introduction Picrorhiza kurroa Royle ex Benth is an important medicinal herb used in the preparation of several herbal drug formulations due to the presence of picroside-I (P-I) and picroside-II (P-II) along with other iridoid-glucosides derivatives. Objective The endangered status of P. kurroa coupled with lack of information on biosynthesis of P-I and P-II necessitate deciphering the biosynthetic pathway for picrosides. Methods LC with electrospray ionisation (ESI) and quadrupole time of flight combined with MS/MS was used to detect intermediates and assemble the picrosides biosynthetic pathway in P. kurroa. Results The presence of catalpol and aucubin, the major backbone structures of picrosides, along with intermediate metabolites boschnaloside, bartsioside and mussaenosidic acid, was confirmed in ESI negative mode with pseudomolecular ion peaks, that is, m/z 361, m/z 343, m/z 345, m/z 329 and m/z 375 ions and their fragmentation patterns. Conclusion The picrosides biosynthetic pathway is expected to provide a reliable platform towards understanding the molecular components (genes/enzymes) of P-I and P-II biosynthesis in P. kurroa for their eventual utilisation in various applications. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 21 May 2013 | 1:28 pm

Chemical and Functional Characterisation of Propolis Collected from East Andalusia (Southern Spain)

Introduction Propolis is a complex mixture of natural sticky, gummy and resinous components produced by honeybees (Apis mellifera L.) from plant materials. However, phytochemical data of the Andalusian (southern Spain) propolis are scant. Objective The primary objectives of this study were to chemically characterise the compounds and evaluate the anti-oxidant activity found in 28 Andalusian propolis samples. Methods Ethanol extracts of propolis (EEP) were prepared and examined for their anti-oxidant activity by 2,2?-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) and 2,2-diphenyl-1-picrylhydrazyl assays. To characterise the phenolic composition, the presence of 11 compounds was identified by HPLC analysis with photodiode array and mass spectrometric detection. Results All propolis samples had strong anti-oxidant activity, accompanied by high total phenolic content. The most abundant compounds were flavonoids. Concerning the phenolic compounds content, our results showed that the 75% of the samples analysed contained at least 80 mg/g of flavonoids, primarily pinobanksin 3-acetate, pinocembrin, chrysin, galangin and pinobanksin. Caffeic acid phenethyl ester was detected in almost all EEP samples but in smaller proportions (mean 12.9?±?2.8 mg/g). Conclusion The present investigation constitutes the first comprehensive report on the phenolics identified in southern Spanish propolis. The results revealed that the samples tested showed a high scavenging activity and therefore indicate the possible use of Andalusian propolis as an important source of natural anti-oxidants. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 14 May 2013 | 7:19 am

Grape Colour Phenotyping: Development of a Method Based on the Reflectance Spectrum

Introduction The colour of fruit is an important quality factor for cultivar classification and phenotyping techniques. Besides the subjective visual evaluation, new instruments and techniques can be used. Objectives This work aims at developping an objective, fast, easy and non-destructive method as a useful support for evaluating grapes' colour under different cultural and environmental conditions, as well as for breeding process and germplasm evaluation, supporting the plant characterization and the biodiversity preservation. Materials and Methods Colours of 120 grape varieties were studied using reflectance spectra. The classification was realized using cluster and discriminant analysis. Reflectance of the whole berries surface was also compared with absorption properties of single skin extracts. Results A phenotyping method based on the reflectance spectra was developed, producing reliable colour classifications. A cultivar-independent index for pigment content evaluation has also been obtained. Conclusions This work allowed the classification of the berry colour using an objective method. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 24 April 2013 | 8:25 am

Analysis of Cleistopholis patens Leaf and Trunk Bark Oils Using Combined GC- Flame Ionisation Detection, GC-Retention Index, GC–MS and 13C-NMR

Introduction Germacrenes A–C are secondary metabolites produced by various plants. They are sesquiterpene hydrocarbons bearing the (E,E)-1,5-cyclodecadiene structure known to undergo thermal rearrangement through a [3.3]-sigmatropic reaction. Such a rearrangement was evidenced by comparing the contents of a given germacrene and the corresponding elemene calculated by GC-flame ionisation detection (FID) with the relative intensities of the signals of both molecules in the 13C-NMR spectrum of the mixture, recorded at room temperature. Objective To develop a protocol to identify and quantify germacrenes A, B and C and in parallel the corresponding elemenes, using a combination of GC-FID and 13C-NMR and then provide a correct analysis of Cleistopholis patens essential oils. Methods The essential oil was submitted to GC-FID, GC-retention index, GC–MS and 13C-NMR analyses. The relative percentages of every couple of germacrene and elemene measured by GC-FID were summed. Then, the relative ratio of the mean intensities of the signals of the protonated carbons of a given germacrene and the corresponding elemene was calculated. The contents of both compounds were obtained by combining GC-FID and 13C-NMR data. Results The true content of germacrene A/?-elemene, germacrene B/?-elemene and germacrene C/?-elemene in leaf and root oils from C. patens was evaluated by combination of GC-FID and 13C-NMR data. Correct analysis of the essential oils was provided. Conclusion Combined analysis of essential oil including 13C-NMR without isolation of the components, appeared really efficient to identify and quantify germacrene isomers and in parallel elemene isomers in essential oils. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 17 April 2013 | 12:12 pm

A Novel Approach in Herbal Quality Control Using Hyperspectral Imaging: Discriminating Between Sceletium tortuosum and Sceletium crassicaule

Introduction Sceletium tortuosum is the most sought after species of the genus Sceletium and is commonly included in commercial products for the treatment of psychiatric conditions and neurodegenerative diseases. However, this species exhibits several morphological and phytochemical similarities to S. crassicaule. Objectives The aim of this investigation was to use ultrahigh-performance liquid chromatography (UPLC) and hyperspectral imaging, in combination with chemometrics, to distinguish between S. tortuosum and S. crassicaule, and to accurately predict the identity of specimens of both species. Methods Chromatographic profiles of S. tortuosum and S. crassicaule specimens were obtained using UPLC with photodiode array detection. A SisuChema near infrared hyperspectral imaging camera was used for acquiring images of the specimens and the data was processed using chemometric computations. Results Chromatographic data for the specimens revealed that both species produce the psychoactive alkaloids that are used as quality control biomarkers. Principal component analysis of the hyperspectral image of reference specimens for the two species yielded two distinct clusters, the one representing S. tortuosum and the other representing S. crassicaule. A partial least squares discriminant analysis model correctly predicted the identity of an external dataset consisting of S. tortuosum or S. crassicaule samples with high accuracy (>94%). Conclusions A combination of hyperspectral imaging and chemometrics offers several advantages over conventional chromatographic profiling when used to distinguish S. tortuosum from S. crassicaule. In addition, the constructed chemometric model can reliably predict the identity of samples of both species from an external dataset. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 17 April 2013 | 12:12 pm

Characterisation of Homoflavonoids from Three Ophioglossum Species Using Liquid Chromatography with Diode Array Detection and Electrospray Ionisation Tandem Mass Spectrometry

Introduction Homoflavonoids, characterised by one more carbon atom directly added to C6–C3–C6 backbone of flavonoids, are rich in the species of genus Ophioglossum. Up to now we have little knowledge about their MS fragmentation patterns. It is therefore necessary to investigate their MS fragmentation pathways so as to distinguish them from other types of flavonoids. Objective To develop a rapid method for identifying homoflavonoids from Ophioglossum based on their characteristic MS fragmentation. Methods Mass spectrometry fragmentation pathways and qualitative analysis of homoflavonoids in three ferns of Ophioglosssum were investigated by using high-performance liquid chromatography coupled with diode-array detection and electrospray ionisation tandem mass spectrometry (HPLC–DAD–ESI/MSn). Results The analyses of the MSn spectra of the homoflavonoids allowed us to classify them into two types according to their fragmentation characteristics. The type I homoflavonoids, with an attached additional carbon atom to the C-3 position of the C-ring, presented the initial competing loss of H2O and CH2O from their aglycone ions, compared to the initial removal of H2O or CO in the case of the type II homoflavonoids, which bear the additional carbon atom at the C-2’ site of the B-ring and forming ring D. The above characteristic fragmentations of homoflavonoids were quite different from those of other flavonoids, and were successfully applied to identify homoflavonoids in the crude extracts of three Ophioglossum species. Conclusion The HPLC–DAD–ESI/MSn method obtained in the present study provided a powerful tool for identifying homoflavonoids from ferns in the genus Ophioglossum. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 12 April 2013 | 4:36 pm

A Simple and Rapid Single Kernel Screening Method to Estimate Amylose Content in Rice Grains

Introduction In rice breeding programmes large number of grain samples are routinely analysed for amylose content (AC) through a tedious spectrophotometric method that also involves high reagent costs. Objective Here, we propose a rapid and economic screening technique for assessment of AC based on the amylose–iodine complex formation in the cut grains of rice, which we refer to as the cut grain dip (CGD) method. Methods The CGD method involves cutting the rice kernels in the middle with a pair of scissors and dipping the cut end in an optimised iodide:iodine (KI-I) solution termed the rapid amylose detection solution (RADS). Results It was found that the time taken for deep blue colouration by the cut end of the grains after dipping in RADS was proportional to the AC. The CGD method was further validated in a large set of rice mutants with varied AC. Conclusion The proposed method can be used to screen samples for AC rapidly, with a single rice caryopsis, without any costly equipment and can be especially suitable for screening of mutants and segregants with altered AC in large breeding populations. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 1 April 2013 | 10:34 am

Ellagic Acid and Derivatives from Cochlospermum angolensis Welw. Extracts: HPLC–DAD–ESI/MSn Profiling, Quantification and In Vitro Anti-depressant, Anti-cholinesterase and Anti-oxidant Activities

Introduction Cochlospermum angolensis Welw. bark is a medicinal plant consumed for the treatment of hepatic diseases and for the prophylaxis of malaria. Nevertheless, there are few studies concerning its chemical composition and biological potential. Objective Since phenolic compounds are described as powerful anti-oxidants and neuroprotective agents, the purpose of this study was to characterise the phenolic profile of this species and to extend the knowledge on its medicinal properties, namely its potential against oxidative stress, Alzheimer's disease and depression. Methods The phenolic composition of aqueous and hydromethanolic extracts was characterised by HPLC–DAD–ESI/MSn. Anti-radical potential was tested against 2,2-diphenyl-1-picrylhdrazyl, superoxide anion and nitric oxide radicals, and neuroprotective effect was assessed against acetylcholinesterase, butyrylcholinesterase and monoamine oxidase A. Results Eight compounds were characterised for the first time. Hydromethanolic extract was richer in methyl ellagic acid and its derivatives, while aqueous extract had higher amounts of ellagic acid and its derivatives. Methyl ellagic acid pentoside isomer and ellagic acid were the major compounds in the two extracts, respectively. Both extracts and ellagic acid revealed radical scavenging capacity stronger than that of ascorbic acid, but a weak effect on cholinesterases was observed. Their anti-depressant activity was also very strong. Conclusion The results provided evidence of the value of C. angolensis as a source of health-promoting anti-oxidants and anti-depressant compounds, with potential to be used as a raw product for food and pharmaceutical industries. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 1 April 2013 | 10:33 am

Quantitative Comparison of Multiple Components in Dioscorea nipponica and D. panthaica by Ultra-High Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry

Introduction Dioscorea nipponica (DN) and D. panthaica (DP) have been uniquely prepared as herbal medicinal products for treating coronary heart disease (CHD) in China. However, so far there has been little discussion and no work comparing the qualitative and quantitative differences between the two herbs nor assessing whether they have similarities in chemical composition that would support their common application for treating CHD. Objective To develop an efficient and reliable method based on UPLC–qTOF–MS for quantitative comparison of saponins in both DN and DP. Methods Using electrospray ionisation and atmospheric-pressure chemical ionisation respectively, six steroidal glycosides and one aglycone were determined in 13 DN samples and 13 DP samples. The comparative analysis of chemical components in DN and DP was carried out by chromatographic fingerprint similarity evaluation, test of significance (t-test) and principle component analysis (PCA). Results The UPLC–qTOF–MS method showed limit of detection and quantitation within the range 0.02–0.2 ng and 0.08–0.5 ng, respectively, for the seven saponins studied. The intra- and interday precision (RSD) were below 5%. The recoveries for the quantified compounds were within the range of 72.79–118.31%. Conclusion This UPLC–qTOF–MS assay provides a suitable method for the identification and determination of major bioactive constituents both in DN and DP. The chemical composition of all DN and DP samples studied exhibited a high level of global similarity. This chemical similarity validates their common application in the pharmaceutical industry as anti-CHD herbal drugs. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 19 March 2013 | 5:10 am

A Rapid Method for Total ?-Escin Analysis in Dry, Hydroalcoholic and Hydroglycolic Extracts of Aesculus hippocastanum L. by Capillary Zone Electrophoresis

Introduction Seeds of Aesculus hippocastanum L. are used in European phytotherapy to treat inflammatory and vascular problems, and also to help in the regulation of the microcirculation. Thus, the quality control of herbal medicines using this species is important. Objective To develop and to optimise a capillary zone electrophoresis method to determine total ?-escin in different extracts of A. hippocastanum L. Methods The optimal condition found through chemometric approach was: 25?mmol/L of bicarbonate–carbonate buffer, pH?10.3; +20?kV of voltage; 20°C of cartridge temperature; direct ultraviolet detection at 226?nm; 13?mbar injection for 5?s and analysis time within 6?min. Results Repeatability, coefficient of variation (CV; %) = 3.19, 3.07 and 1.89 (n = 12), and intermediate precision, CV (%) = 3.05, 3.53 and 2.99 (n = 24) for dry, hydroalcoholic and hydroglycolic extracts, respectively were achieved. The accuracy was evaluated through recovery tests in concentration levels of 100, 150 and 200 g/L, ranging from 98.17 to 104.68%. The proposed method exhibited linearity (r?=?0.9983) in the concentration range from 101.4 to 907.2?g/L and limits of detection and quantification equal to 11.63 and 38.76?g/L respectively. Conclusion A fast and reliable methodology for determination of total ?-escin was successfully validated and applied on extracts of A. hippocastanum L. demonstrating its usefulness to quality control of medicines containing this plant species. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 19 March 2013 | 5:10 am

Qualitative and Quantitative Analysis of Phenolic Compounds in the Leaves of Aquilaria sinensis Using Liquid Chromatography–Mass Spectrometry

Introduction The leaves of Aquilaria sinensis traditionally have been used in China for a century. Phenolic compounds were investigated to be the major active compounds in them. Objective To establish a method that will simultaneously determine the phenolic compounds in Aquilaria sinensis leaves and identify their characteristic fragmentation patterns, and to make a comparison of A. sinensis leaf samples from different areas of China. Methods High-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) and photodiode array detection (DAD) were used for the qualitative and quantitative analysis. Results Twenty-one compounds, including xanthones, benzophenones and flavones, were identified or tentatively characterised. The fragmentation patterns of xanthones and benzophenones were also described. Also, eight components in the herbal samples from different regions were determined by HPLC-DAD. Conclusion The method developed is suitable for the qualitative and quantitative analysis of phenolic constituents in the leaves of A. sinensis. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 11 March 2013 | 3:07 am

A Modern Approach to the Authentication and Quality Assessment of Thyme Using UV Spectroscopy and Chemometric Analysis

Introduction Recently, the fields of chemometrics and multivariate analysis have been widely implemented in the quality control of herbal drugs to produce precise results, which is crucial in the field of medicine. Thyme represents an essential medicinal herb that is constantly adulterated due to its resemblance to many other plants with similar organoleptic properties. Objective To establish a simple model for the quality assessment of Thymus species using UV spectroscopy together with known chemometric techniques. The success of this model may also serve as a technique for the quality control of other herbal drugs. Materials and methods The model was constructed using 30 samples of authenticated Thymus vulgaris and challenged with 20 samples of different botanical origins. The methanolic extracts of all samples were assessed using UV spectroscopy together with chemometric techniques: principal component analysis (PCA), soft independent modeling of class analogy (SIMCA) and hierarchical cluster analysis (HCA). Results The model was able to discriminate T. vulgaris from other Thymus, Satureja, Origanum, Plectranthus and Eriocephalus species, all traded in the Egyptian market as different types of thyme. The model was also able to classify closely related species in clusters using PCA and HCA. The model was finally used to classify 12 commercial thyme varieties into clusters of species incorporated in the model as thyme or non-thyme. Conclusion The model constructed is highly recommended as a simple and efficient method for distinguishing T. vulgaris from other related species as well as the classification of marketed herbs as thyme or non-thyme. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 11 March 2013 | 12:59 am

Interval Multivariate Curve Resolution in the Dereplication of HPLC–DAD Data from Jatropha gossypifolia

Introduction Jatropha gossypifolia has been used quite extensively by traditional medicine for the treatment of several diseases in South America and Africa. This medicinal plant has therapeutic potential as a phytomedicine and therefore the establishment of innovative analytical methods to characterise their active components is crucial to the future development of a quality product. Objective To enhance the chromatographic resolution of HPLC–UV–diode-array detector (DAD) experiments applying chemometric tools. Methods Crude leave extracts from J. gossypifolia were analysed by HPLC–DAD. A chromatographic band deconvolution method was designed and applied using interval multivariate curve resolution by alternating least squares (MCR–ALS). Results The MCR–ALS method allowed the deconvolution from up to 117% more bands, compared with the original HPLC–DAD experiments, even in regions where the UV spectra showed high similarity. The method assisted in the dereplication of three C-glycosylflavones isomers: vitexin/isovitexin, orientin/homorientin and schaftoside/isoschaftoside. Conclusion The MCR–ALS method is shown to be a powerful tool to solve problems of chromatographic band overlapping from complex mixtures such as natural crude samples. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 11 March 2013 | 12:59 am

Determination of Endogenous Brassinosteroids in Plant Tissues Using Solid-phase Extraction with Double Layered Cartridge Followed by High-performance Liquid Chromatography–Tandem Mass Spectrometry

Introduction Brassinosteroids (BRs) are a group of important phytohormones that play vital roles in plant growth, development and a series of physiological phenomena. In order to understand biosynthesis, degradation and metabolic pathways of BRs, a reliable analytical method of BRs with effective sample pre-treatment process is favourable. Objective The development of a quick and effective method for the quantification of endogenous BRs in plant tissue with the aid of double layered solid-phase extraction (SPE) cartridges (graphite carbon black and primary secondary amine silica sorbent: GCB/PSA). Method The method involved an initial extraction of BRs with acetonitrile, a dehydration process with anhydrous MgSO4 and NaCl, a SPE purification process with a double layered cartridge, and a further clean-up step utilising liquid–liquid extraction (LLE). The purification process was mainly realised on the GCB/PSA cartridge. GCB could eliminate hydrophobic compounds, especially those containing a ? system, and PSA was introduced to remove the polar interferences. Endogenous BRs were quantified by HPLC–ESI–MS/MS. Results Good linearities were obtained in the range of 0.4–500?ng/mL (0.0124–15?ng), with the correlation coefficients above 0.9957. The relative recoveries of BRs of this method were in the range of 71.1–113.1%, with intra- and interday relative standard deviations (RSDs) less than 16.3%. With the proposed method, the requirement of plant tissue amount was minimised to 1?g fresh weight, which is the smallest amount reported so far, to our knowledge. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 25 February 2013 | 7:05 am

Combination of Bioautography with HPTLC–MS/NMR: A Fast Identification of Acetylcholinesterase Inhibitors from Galbanum

Introduction In the search for new natural compounds with acetylcholinesterase (AChE) inhibitory activity this study focused on galbanum, the oleo gum-resin from Ferula gummosa Boiss., which had shown AChE inhibitory activity in a screening. Objective The isolation of bioactive compounds from plant extracts usually is laborious and time consuming. In an approach to accelerate the characterisation of compounds with AChE inhibitory activity, the potential of a combination of HPTLC bioautography with HPTLC–MS/NMR for the fast identification of active compounds in galbanum was studied. Method Pre-fractionation of the dichloromethane extract was performed by vacuum liquid chromatography. The resulting fractions were separated by HPTLC and active zones determined by bioautography. A TLC–MS interface was used to elute the single zones from the plates directly into a mass spectrometer. The interface was also used to extract the two major active zones from HPTLC plates for off-line one- and two-dimensional NMR and quadrupole time of flight (QTOF) MS. Results The isolated compounds were identified as 7-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}-2H-chromen-2-one (auraptene) and 7-{[(1R,4aR,6S,8aS)-6-hydroxy-5,5,8a-trimethyl-2-methylenedecahydronaphthalen-1-yl]methoxy}-2H-chromen-2-one (farnesiferol A). This is the first report of these substances in F. gummosa. Their median inhibitory concentration (IC50) values for AChE inhibition were determined as 47 and 17 µg/mL in comparison with physostigmine as a positive control (IC50: 0.8 µg/mL) and their concentrations in galbanum were quantified by HPLC as 3.5% and 7.9%, respectively. Conclusion The study showed that HPTLC–MS/NMR can be considered as a fast and high-confidence method for dereplication of natural compounds. From the correlation of the concentration of the elucidated compounds and their IC50 values for AChE inhibition it can be concluded that auraptene and farnesiferol A are contributing to this activity of galbanum. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 21 February 2013 | 1:15 pm

Simultaneous Determination of Five Bioactive Components in Radix Glycyrrhizae by Pressurised Liquid Extraction Combined with UPLC–PDA and UPLC/ESI–QTOF–MS Confirmation

Introduction Glycyrrhizae species are popular ingredients of herbal medicine in most traditional Chinese medicine prescriptions, and they mainly contain flavonoids and triterpene saponins. The contents of these bioactive compounds may vary in different batches and affect the therapeutic effects. Thus comprehensive quality control and monitoring of their herbal formulation are of paramount concern. Objective To establish a rapid, effective pressurised liquid extraction (PLE) and ultra-performance liquid chromatography coupled with photodiode array (UPLC–PDA) method to evaluate the quality of Glycyrrhizae species. Methods Radix Glycyrrhizae was extracted by PLE using 70% ethanol at 100°C for 15 min during three static extraction cycles. Separation was performed using an UPLC system to quantify five bioactive compounds, namely liquiritin apioside, liquiritin, liquiritigenin, glycyrrhizic acid and glycyrrhetinic acid, in 12 batches of samples of different origins in China. Furthermore, the samples were analysed using an ultra-performance liquid chromatography coupled with electrospray ionisation and time-of-flight mass spectrometry (UPLC/ESI–QTOF–MS) system to confirm the results. Results The calibration curves of all five analytes showed good linearity (R2?>?0.9997). Accuracy, precision and repeatability were all within required limits. The mean recoveries measured at the three concentrations were higher than 93.7% with RSDs lower than?<?3.33% for the targets. Conclusion The established PLE and UPLC–PDA method could serve as a rapid and effective method for quality evaluation of Radix Glycyrrhizae. The UPLC technique can be considered as an attractive alternative to HPLC in routine quality control of Chinese medicine, especially in situations where high sample throughput and fast analytical speed are required. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 21 February 2013 | 12:28 pm

Analyses of the Iridoid Glucoside Dimers in Paederia scandens Using HPLC–ESI–MS/MS

Introduction Many dimers consisting of structurally similar monomers are difficult to be identified even using NMR. Rapid structural identification of iridoid glycoside dimers, especially isomeric dimers in a complicated matrix, remains desirable. Objective To develop a rapid, sensitive analytical method for structural elucidation of trace iridoid glycoside isomers in a complicated extract. Methods Three isomeric iridoid glucoside dimers, paederoscandoside, saprosmoside E and saprosmoside D, were isolated and further analysed by electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (ESI–QTOF–MS/MS) in positive-ion mode. Energy-resolved mass spectrometry (ERMS) was used to provide information on the relative intensity of ions versus collision energy. The crude extract of Paederia scandens was analysed by HPLC–ESI–MS/MS. Results The relative abundance of product ions in the MS/MS spectra from ammonium adduct ions varied greatly for the three isomers. The energy-resolved experiments enhanced differences among the three isomers. A total of 13 iridoid glucoside dimers (three groups of isomers) in the extract of P. scandens were identified or tentatively characterised by using HPLC–ESI–QTOF based on the tandem mass spectra of references. Conclusion Linkage sites between different hydroxyl groups on the sugar and carboxyl groups for the three groups of isomers are confirmed. The reason for fragmentation differences might be that cleavage of the glycosidic bond accompanies the active H in vicinal hydroxyl rearrangement. The MS method is a useful tool for the analysis of isomers, especially trace isomers in a complicated extract. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 21 February 2013 | 12:06 pm

Stepwise Elution of a Three-phase Solvent System in Centrifugal Partition Extraction: A New Strategy for the Fractionation and Phytochemical Screening of a Crude Bark Extract

Introduction Tree bark represents an interesting source of bioactive molecules for the discovery of new pharmaceutical agents. However, the detailed screening of secondary metabolites in crude bark extracts is often hampered by the presence of tannins, which are difficult to separate from other plant constituents. Objective In the present study, a new centrifugal partition extraction (CPE) method was developed in order to fractionate a crude bark extract of Anogeissus leiocarpus Guill. & Perr. (Combretaceae). Methods A three-phase solvent system composed of n-heptane, methyl tert-butyl ether, acetonitrile and water was optimised for the stepwise elution at 20 mL/min of different phytochemical classes according to their hydrophobicity. Onedimensional and two-dimensional NMR analyses of the simplified fractions were then performed in order to characterise potentially interesting metabolites. Results In one step, 5 g of the initial crude extract were efficiently fractionated to yield highly simplified fractions that contained triterpenes, ellagic acid derivatives, flavonoids and phenolic compounds. All undesired compounds, that is, the highly abundant water-soluble tannins (78.8%), were totally removed and each run was rapidly achieved in 90 min on a the multi-gram scale and with low solvent volumes. Conclusion Centrifugal partition extraction in the elution mode using a three-phase solvent system can thus be proposed as an efficient and cost-effective alternative for a rapid fractionation of crude bark extracts and for an effective screening of potentially active secondary metabolites. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 21 February 2013 | 11:58 am

Assessment of Phytoestrogen and Mycoestrogen Recognition by Recombinant Human Estrogen Receptor-? Using Ligand Titration Arrays

Introduction Exposure to phytoestrogens and mycoestrogens has emerged as a public health issue due to their potentially endocrine disruption activities resulting from direct interaction with sex-steroid hormone receptors. There is a significant requirement for comprehensive, reproducible methods to determine the extent of estrogen mimicry by compounds encountered in the environment to estimate risk:benefit ratios, particularly in humans. Objective To develop a systematic approach for assessing recognition of chemically diverse compounds by human estrogen receptor proteins to aid in their assessment as endocrine disruptor compounds (EDCs). Methods Recombinant human estrogen receptor-? protein (rhER?) was expressed in Saccharomyces cervisiae as an ubiquitin fusion under control of a CUP1 promoter and partially purified with heparin affinity chromatography in the unliganded state. A novel radio-ligand binding array was developed to evaluate structurally diverse compounds, both naturally occurring and synthetic, for estrogen binding activity and affinity. Results Binding affinities of suspected estrogen mimics for rhER? were calculated over a range of [3H]estradiol-17? concentrations using Lundon OneSite® and Compete® software. Conclusion ?-zearalanol, a mycoestrogen similar to zearalenone used as an ICCVAM validation substance for the in vitro estrogen receptor binding assays (ICCAM report), was employed as a model estrogen mimic to illustrate the approach, methods and calculations using these techniques. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 9 February 2013 | 5:55 am

A New Application of Charged Aerosol Detection in Liquid Chromatography for the Simultaneous Determination of Polar and Less Polar Ginsenosides in Ginseng Products

Introduction Conventional liquid chromatographic methods coupled with ultraviolet or evaporative light scattering detection are not sensitive enough to determine both polar and less polar ginsenosides at low concentrations. Objective To establish a liquid chromatography–charged aerosol detection method for the simultaneous determination of polar and less polar ginsenosides in a variety of ginseng products Methods Fourteen polar and less polar ginsenosides were extracted and concentrated by solid phase extraction. These were subsequently baseline-separated on a conventional reversed-phase C18-column (250?mm?×?4.6?mm, 5?µm) with a simple mobile phase consisting of water and acetonitrile. Components were then detected by means of charged aerosol detection. Results The method developed allowed the simultaneous determination of six polar ginsenosides (Rg1, Re, Rb1, Rc, Rb, Rd) and eight less polar ginsenosides (Rg6, F4, Rk3, Rh4, Rg3(S), Rg3(R), Rk1, Rg5) in a single chromatographic run. Further, the method was linear (R2?>?0.99), accurate (relative recoveries, 90–112%), and precise (intraday RSD?<?5.7% and interday RSD?<?10.6%) within the concentration range tested. The method sensitivity was measured in terms of the limit of detection, which ranged from 0.5 to 4.0?µg/mL. Conclusion Concentrations of 14 ginsenosides were determined simultaneously in one homemade red ginseng and 13 commercial ginseng products of different types (liquid and solid samples), and results showed that ginsenoside content varied significantly among the samples tested. The method developed could serve as a useful analytical tool for the quality control of ginseng products. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 7 February 2013 | 8:50 am

Developmental Changes in the Composition of Five Anthraquinones from Rheum palmatum as Quantified by 1H-NMR

Introduction Rheum palmatum is an important traditional Chinese medicine featuring anthraquinones with several activities. Generally, rhein, emodin, aloe-emodin, physcion and chrysophanol are used as chemical markers for the quality control of rhubarb products. Objective To develop a simple protocol for the quantification of rhein, emodin, aloe-emodin, physcion and chrysophanol in R. palmatum collected at different developmental stages. Methods 1H-NMR spectra were measured on samples dissolved in acetone-d6, quantification was carried out using the signals of H-4 of rhein (?H 8.36), H-7 of emodin (?H 6.68), CH2OH of aloe-emodin (?H 4.81), OCH3 of physcion (?H 4.02) and CH3 of chrysophanol (?H 2.50), which were well separated from other signals. Quantitative analysis was based on the relative ratio of the intensity of each compound to the known amount of internal standard maleic acid. Results The quantitative 1H-NMR (qHNMR) method developed showed good precision, trueness, linearity, repeatability and stability for the quantification of rhein, emodin, aloe-emodin, physcion and chrysophanol. This method was applied successfully to explore the seasonal variations of the five major anthraquinones in R. palmatum, and provided quantitative results in reasonable agreement with those obtained by the HPLC–UV method. Conclusion Compared with the conventional HPLC-based methods, the qHNMR analysis is rapid, reference-free and convenient with less sample pre-treatment. This technique should be a feasible choice for the quality control of R. palmatum. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 31 January 2013 | 1:25 pm

Analysis of Mogroside V in Siraitia grosvenorii with Micelle-mediated Cloud-Point Extraction

Introduction Mogroside V is the main effective ingredient of Siraitia grosvenorii used as a natural sweet food as well as a traditional Chinese medicine. The sample pre-treatment prior to chromatographic analysis requires large amounts of toxic organic solvents and is time consuming. Objective To develop an effective and simple method for extracting and determining mogroside V of Siraitia grosvenorii. Methods Mogroside V was extracted and preconcentrated by micelle-mediated cloud-point extraction with nonionic surfactant isotridecyl poly (ethylene glycol) ether (Genapol® X-080). The obtained solutions containing mogroside V were analysed by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The chromatographic separation was performed on a C18-column using gradient elution with acetonitrile and water at 203?nm. Results The cloud-point extraction yield was 80.7% while the pre-concentration factor was about 10.8. The limit of detection was 0.75?µg/mL and the limit of quantification was 2?µg/mL. The relative standard deviations for intra- and interday precisions of mogroside V were less than 8.68% and 5.78%, respectively, and the recoveries were between 85.1% and 103.6%. Conclusion The HPLC–UV method based on micelle-mediated cloud-point extraction for determination mogroside V in Siraitia grosvenorii was environmentally friendly, simple and sensitive. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 25 January 2013 | 4:20 am

In-tube Extraction and GC–MS Analysis of Volatile Components from Wild and Cultivated sea buckthorn (Hippophae rhamnoides L. ssp. Carpatica) Berry Varieties and Juice

Introduction The health benefits of sea buckthorn (Hippophae rhamnoides L.) are well documented due to its rich content in bioactive phytochemicals (pigments, phenolics and vitamins) as well as volatiles responsible for specific flavours and bacteriostatic action. The volatile compounds are good biomarkers of berry freshness, quality and authenticity. Objective To develop a fast and efficient GC–MS method including a minimal sample preparation technique (in-tube extraction, ITEX) for the discrimination of sea buckthorn varieties based on their chromatographic volatile fingerprint. Material and methods Twelve sea buckthorn varieties (wild and cultivated) were collected from forestry departments and experimental fields, respectively. The extraction of volatile compounds was performed using the ITEX technique whereas separation and identification was performed using a GC–MS QP-2010. Principal component analysis (PCA) was applied to discriminate the differences among sample composition. Results Using GC–MS analysis, from the headspace of sea buckthorn samples, 46 volatile compounds were separated with 43 being identified. The most abundant derivatives were ethyl esters of 2-methylbutanoic acid, 3-methylbutanoic acid, hexanoic acid, octanoic acid and butanoic acid, as well as 3-methylbutyl 3-methylbutanoate, 3-methylbutyl 2-methylbutanoate and benzoic acid ethyl ester (over 80% of all volatile compounds). Principal component analysis showed that the first two components explain 79% of data variance, demonstrating a good discrimination between samples. Conclusion A reliable, fast and eco-friendly ITEX/GC–MS method was applied to fingerprint the volatile profile and to discriminate between wild and cultivated sea buckthorn berries originating from the Carpathians, with relevance to food science and technology. Copyright © 2013 John Wiley & Sons, Ltd.

Posted on 15 January 2013 | 10:36 am

C-glucosidic Ellagitannins from Lythri herba (European Pharmacopoeia): Chromatographic Profile and Structure Determination

Introduction Lythri herba, a pharmacopoeial plant material (European Pharmacopoea), is obtained from flowering parts of purple loosestrife (Lythrum salicaria L.). Although extracts from this plant material have been proven to possess some interesting biological activities and its pharmacopoeial standardisation is based on total tannin content determination, the phytochemical characterisation of this main group of compounds has not yet been fully conducted. Objective To isolate ellagitannins from Lythri herba, determine their structures and develop chromatographic methods for their qualitative analysis. Results Five C-glucosidic ellagitannins – monomeric- vescalagin and castalagin together with new dimeric structures – salicarinins A–C, composed of vescalagin and stachyurin, vescalagin and casuarinin, castalagin and casuarinin units connected via formation of valoneoyl group, were isolated using column chromatography and preparative HPLC. Structures were determined according to 1H and 13C-NMR (one- and two-dimensional), electrospray ionisation–time of flight (ESI–TOF), electrospray ionisation-ion trap (ESI-MSn) and circular dichroism (CD) spectra, together with acidic hydrolysis products analysis. HPTLC on RP-18 modified plates and HPLC–DAD–MSn on RP-18 column methods were developed for separation of the five main ellagitannins. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 28 December 2012 | 10:28 am

UPLC–Q-TOF–HDMS Analysis of Constituents in the Root of Two Kinds of Aconitum Using a Metabolomics Approach

Introduction Metabolomics is an 'omics' approach that aims to comprehensively analyse all metabolites in a biological sample, and has great potential for directly elucidating plant metabolic processes. Increasing evidence supports the view that plants produce a broad range of low-molecular-weight secondary metabolites responsible for variation from species to species, thus enabling the use of secondary metabolite profiling in the chemotaxonomy. Objective To gain deeper insights into the metabolites to increasing plant diversity, we performed systematic untargeted metabolite profiling to exploit the different parts and species of Aconitum as a case study. Method Application of ultraperformance liquid chromatography–quadrupole time-of-flight–high-definition mass spectrometry (UPLC–QTOF–HDMS) equipped with electrospray ionisation and coupled with pattern recognition analyses to study constituents in the root of two kinds of Aconitum species. Results Twenty-two metabolites between the mother root of Aconitum carmichaelii Debx (CHW) and lateral root of Aconitum carmichaelii Debx (SFZ) and 13 metabolites between the CHW and root of Aconitum kusnezoffii Reichb (CW) have been identified. Of note, songorine, carmichaeline and isotalatizidine did not exist in CW, whereas they are present in the SFZ and CHW. Conclusion Metabolomics based UPLC–QTOF–HDMS with multivariate statistical models was effective for analysis of constituents in the root of two kinds of Aconitum species. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 7 December 2012 | 1:48 am

Quantification of Phenylpropanoids in Commercial Echinacea Products Using TLC with Video Densitometry as Detection Technique and ANN for Data Modelling

Introduction Echinacea preparations are among the most popular herbal remedies worldwide. Although it is generally assigned immune enhancement activities, the effectiveness of Echinacea is highly dependent on the Echinacea species, part of the plant used, the age of the plant, its location and the method of extraction. Objective The aim of this study was to investigate the capacity of an artificial neural network (ANN) to analyse thin-layer chromatography (TLC) chromatograms as fingerprint patterns for quantitative estimation of three phenylpropanoid markers (chicoric acid, chlorogenic acid and echinacoside) in commercial Echinacea products. Material and methods By applying samples with different weight ratios of marker compounds to the system, a database of chromatograms was constructed. One hundred and one signal intensities in each of the TLC chromatograms were correlated to the amounts of applied echinacoside, chlorogenic acid and chicoric acid using an ANN. Results The developed ANN correlation was used to quantify the amounts of three marker compounds in Echinacea commercial formulations. The minimum quantifiable level of 63, 154 and 98?ng and the limit of detection of 19, 46 and 29?ng were established for echinacoside, chlorogenic acid and chicoric acid respectively. Conclusion A novel method for quality control of herbal products, based on TLC separation, high-resolution digital plate imaging and ANN data analysis has been developed. The method proposed can be adopted for routine evaluation of the phytochemical variability in Echinacea formulations available in the market. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 22 November 2012 | 9:29 am

Phenolic Profiling of Portuguese Propolis by LC–MS Spectrometry: Uncommon Propolis Rich in Flavonoid Glycosides

Introduction Propolis is a chemically complex resinous substance collected by honeybees (Apis mellifera) from tree buds, comprising plant exudates, secreted substances from bee metabolism, pollen and waxes. Its chemical composition depends strongly on the plant sources available around the beehive, which have a direct impact in the quality and bioactivity of the propolis. Being as Portugal is a country of botanical diversity, the phenolic characterisation of propolis from the different regions is a priority. Objective Extensive characterisation of the phenolic composition of Portuguese propolis from different continental regions and islands. Method Forty propolis ethanolic extracts were analysed extensively by liquid chromatography with diode-array detection coupled to electrospray ionisation tandem mass spectrometry (LC–DAD–ESI–MSn). Results Seventy-six polyphenols were detected in the samples and two groups of propolis were established: the common temperate propolis, which contained the typical poplar phenolic compounds such as flavonoids and their methylated/esterified forms, phenylpropanoid acids and their esters, and an uncommon propolis type with an unusual composition in quercetin and kaempferol glycosides – some of them never described in propolis. Conclusion The method allowed the establishment of the phenolic profile of Portuguese propolis from different geographical locations, and the possibility to use some phenolic compounds, such as kaempferol-dimethylether, as geographical markers. Data suggest that other botanical species in addition to poplar trees can be important sources of resins for Portuguese propolis. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 22 November 2012 | 9:13 am

Influence of Bracken Fern (Pteridium caudatum L. Maxon) Pre-treatment on Extraction Yield of Illudane Glycosides and Pterosins

Introduction Bracken (Pteridium spp) illudane glycosidess are labile biologically active terpenoids that undergo decomposition in mild alkali or acid, heat and enzymatic reactions. Hypothetically, quantitation of these weakly chromophoric carcinogens may be challenged by plant sample preparation procedures that may alter the yield of isolates. Objective To study the influence of common plant sample pre-treatments on the recovery of Pteridium caudatum illudane glycoside carcinogens, ptaquiloside (1a), caudatoside (1c) and ptaquiloside Z (1d), and associated pterosins A, B and Z (2a, b, c) using HPLC–DAD. Method Bracken fronds were divided in equal left/right sections. One section was subjected to high vacuum desiccation (VD) and the other to freeze-drying (FD), air drying at room temperature (AD) for 7?days, air drying at 70?°C for 72?h (HD), or no treatment (fresh frond, FF). Quantitation was achieved by brief hot-water extraction, base-acid transformation of 1a, 1c and 1d to 2a, b, c and HPLC–DAD analysis against standards. Results Substantial differences in extraction yields were found for all illudane glycosides in the order FF?>?FD???VD?>?AD?>?HD. Illudane instability to HD was 1c?>?1d?>?1a. Significant losses also were recorded in yields of Pterosins A, B and Z. Conclusion Glycoside extraction suffers from substantial yield loss of all illudane glycosides and indigenous pterosins in all sample pre-treatments studied relative to fresh frond material. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 20 November 2012 | 2:15 pm

Ultrasonic Nebulisation Extraction: Extraction Column Coupled with Liquid Phase Microextraction for Analysis of the Volatile Organic Compounds in Foeniculum vulgare Mill. as a Model

Introduction As the concentrations of the volatile organic compounds are always low and their matrix is complex, it is necessary to pre-concentrate the volatile organic compounds before analysis. Ultrasonic nebulisation extraction with a self-made extraction column coupled with liquid phase microextraction is developed for the extraction of active constituents from spices. Objective To develop an environmentally compatible extraction technique for the preparation and analysis of the volatile organic compounds from spices. Method The sample is placed into the nebulisation vessel of a nebulisation humidifier and a purging gas is blown through the vessel continuously. When the nebuliser is switched on, a ultrasonic fountain is formed by ultrasonic vibration and the target analytes are transferred from the sample solution to the vapour phase and then concentrated on the extraction solvent in the extraction column. After extraction for 3?min and allowed to stand upright for 5?min, the extract is analysed by GC and GC–MS. Different methods of comparison can then be carried out. Results Optimum conditions were found to be: 30 ?L of n-tetradecane as the extraction solvent, a flow rate for the purging gas of 40?mL/min, a purging time of 3?min and a standing time was 5?min. The contents of constituents in the extract obtained by the proposed method were close to those obtained by hydrodistillation (HD). Moreover, the proposed method achieves higher enrichment efficiency. Conclusion A method was developed for the extraction of volatile organic compounds from spices. The study has shown that it is a fast and environmentally sustainable technique. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 25 October 2012 | 2:55 pm

Production of Polyclonal Antibody Against Madecassoside and Development of Immunoassay Methods for Analysis of Triterpene Glycosides in Centella asiatica

Introduction Centella asiatica (L.) Urban consists of two major triterpene glycosides, asiaticoside (AS) and madecassoside (MA), as active components used for wound healing and enhancing memory. Objective To produce a polyclonal antibody against madecassoside (MA-PAb) and develop enzyme-linked immunosorbent assay (ELISA) and Eastern blotting methods for quantitative analysis of triterpene glycosides in Centella asiatica. Methods An ELISA method was developed using polyclonal antibody against MA. An Eastern blotting method on the PES membrane was established for determination of MA and AS. The immunoassays were validated for sensitivity, precision, specificity and accuracy. Results The prepared MA-PAb shows specificity to MA and AS. The measuring range of triterpene glycosides was 0.39–50?µg/mL using the ELISA method. An Eastern blotting method was developed for determining individual MA and AS, which could be detected in the range of 62.5–500?ng. The limit of detection for MA and AS was 31.25?ng. The two methods developed showed good specificity, precision, and accuracy, and also correlated with high-performance liquid chromatography. Conclusion These immunoassays have several advantages that include high sensitivity as well as being rapid and facile for determination of the triterpene glycosides in C. asiatica. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 19 October 2012 | 11:23 am

Design of Experiment Approach for the Process Optimisation of Microwave Assisted Extraction of Lupeol from Ficus racemosa Leaves Using Response Surface Methodology

Introduction Triterpenoids are a group of important phytocomponents from Ficus racemosa (syn. Ficus glomerata Roxb.) that are known to possess diverse pharmacological activities and which have prompted the development of various extraction techniques and strategies for its better utilisation. Objective To develop an effective, rapid and ecofriendly microwave-assisted extraction (MAE) strategy to optimise the extraction of a potent bioactive triterpenoid compound, lupeol, from young leaves of Ficus racemosa using response surface methodology (RSM) for industrial scale-up. Material and Method Initially a Plackett–Burman design matrix was applied to identify the most significant extraction variables amongst microwave power, irradiation time, particle size, solvent:sample ratio loading, varying solvent strength and pre-leaching time on lupeol extraction. Among the six variables tested, microwave power, irradiation time and solvent–sample/loading ratio were found to have a significant effect (P?<?0.05) on lupeol extraction and were fitted to a Box–Behnken-design-generated quadratic polynomial equation to predict optimal extraction conditions as well as to locate operability regions with maximum yield. Results The optimal conditions were microwave power of 65.67% of 700?W, extraction time of 4.27?min and solvent–sample ratio loading of 21.33?mL/g. Confirmation trials under the optimal conditions gave an experimental yield (18.52?µg/g of dry leaves) close to the RSM predicted value of 18.71?µg/g. Conclusion Under the optimal conditions the mathematical model was found to be well fitted with the experimental data. The MAE was found to be a more rapid, convenient and appropriate extraction method, with a higher yield and lower solvent consumption when compared with conventional extraction techniques. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 15 October 2012 | 2:50 pm

Dehydropyrrolizidine Alkaloids in Two Cryptantha Species: Including Two New Open Chain Diesters One of Which is Amphoteric

Introduction A livestock poisoning outbreak near Kingman, Arizona, USA, potentially linked to dehydropyrrolizidine alkaloids, prompted an evaluation of some local plants for the presence of these hepatotoxic alkaloids. Objective To qualitatively and quantitatively examine two species of Cryptantha, a Boraginaceous genus previously shown to produce potentially toxic pyrrolizidine alkaloids, collected from the vicinity of Kingman, Arizona. Method Plant extracts were analysed using HPLC–electrospray ionisation (+)–MS and MS/MS to determine the presence of dehydropyrrolizidine alkaloid esters. Identities were confirmed by comparison of chromatographic and MS data with authenticated standards and, in the case of the previously undescribed alkaloids, using one- and two-dimensional NMR spectroscopy and high-resolution mass measurement. Results Cryptantha inequata and C. utahensis were shown to produce retronecine-based dehydropyrrolizidine alkaloids at approximately 0.05% and 0.09% w/w respectively. Cryptantha inequata produced mainly echimidine, acetylechimidine and echiuplatine; dehydropyrrolizidine alkaloids that were previously associated with Echium plantagineum. The previously undescribed structure of echiuplatine was elucidated as an amphoteric, open chain diester with angelic acid and 3-hydroxy-3-methylglutaric acid. Along with lycopsamine, intermedine and dihydroxyechiumine, C. utahensis produced cryptanthine, a previously undescribed open chain diester alkaloid esterified with angelic acid and 2,3-dihydroxy-2-methylbutanoic acid. All pyrrolizidine alkaloids detected were present in the plants mainly as their N-oxides. Conclusion The retronecine-based alkaloids detected in both Cryptantha species herein investigated aligns them within the Krynitzkia subgenus. The dehydropyrrolizidine alkaloids detected are expected to be toxic but the low levels in the plants potentially mitigate the risk. The identification of the amphoteric echiuplatine provides a cautionary note with respect to the analysis of total dehydropyrrolizidine alkaloid content. Published 2012. This article is a US Government work and is in the public domain in the USA.

Posted on 15 October 2012 | 2:48 pm

Metabolome Classification of Brassica napus L. Organs via UPLC–QTOF–PDA–MS and Their Anti-oxidant Potential

Introduction Brassica napus L. is a crop widely grown for its oil production and other nutritional components in the seed. In addition to the seed, other organs contain a wide range of phenolic metabolites although they have not been investigated to the same extent as in seeds. Objective To define and compare the phytochemical composition of B. napus L. organs, namely the root, stem, leaf, inflorescence and seeds. Method Non-targeted metabolomic analysis via UPLC–QTOF–MS was utilised in order to localise compounds belonging to various chemical classes (i.e. oxygenated fatty acids, flavonols, phenolic acids and sinapoyl choline derivatives). Results The vast majority of identified metabolites were flavonol glycosides that accumulated in most of the plant organs. Whereas other classes were detected predominantly in specific organs, i.e. sinapoyl cholines were present uniquely in seeds. Furthermore, variation in the accumulation pattern of metabolites from the same class was observed, particularly in the case of quercetin, kaempferol and isorhamnetin flavonols. Anti-oxidant activity, based on 2,2-diphenyl-1-picrylhdrazyl analysis was observed for all extracts, and correlated to some extent with total flavonoid content. Conclusion This study provides the most complete map for polyphenol composition in B. napus L. organs. By describing the metabolites profile in B. napus L., this study provides the basis for future investigations of seeds for potential health and/or medicinal use. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 11 October 2012 | 10:20 am

Improvements in the Separation Capabilities of Sequential Injection Chromatography: Determination of Intracellular Dissolved Free Amino Acid Profiles in Three Taxonomic Groups of Microalgae

Introduction Dissolved free amino acids (DFAA) in intracellular extracts of marine microalgae can be determined by sequential injection chromatography (SIC). This technique uses portable, low-cost instrumentation but its applications have been limited to short monolithic columns because of components not resistant to high pressures. Objective To develop a SIC method for determination of DFAA by exploring an instrument modified to handle pressures of 1000?psi. Method The method was based on pre-column derivatisation of the amino acids with o-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 9.4), separation and fluorimetric detection (?excitation?=?340 and ?emission?=?450?nm). Separation was achieved by stepwise gradient elution using six mobile phases. The first elution step used a mobile phase composed of methanol:tetrahydrofuran:10?mm phosphate buffer (pH 7.2) at a volumetric ratio of 8:1:91. Additional elution steps used mobile phases containing methanol and 10?mM phosphate buffer at ratios of 17.5:82.5, 25:75, 35:65, 50:50 and 65:35. Results Nineteen chromatographic peaks were observed in a mixture of 20 amino acids. The only complete co-elution was between tryptophan and methionine. Detection limits varied from 0.10?µm for isoleucine to 1.5?µm for lysine. Recoveries from spiked extracts were between 84 and 131%. Conclusion Resolutions of the amino acid pairs glutamine and histidine, valine and phenylalanine, and isoleucine and leucine were 1.5, 0.75 and 1.3, respectively. The proposed method found different profiles of DFAA among the three species of algae, suggesting its adequacy for metabolic studies. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 11 October 2012 | 10:13 am

HPLC Determination of the Major Active Flavonoids and GC–MS Analysis of Volatile Components of Dysphania graveolens (Amaranthaceae)

Introduction Dysphania graveolens is used mainly in Mexican traditional medicine against gastrointestinal ailments. Previous investigations revealed that its flavonoids are important active principles; however, there is not a reliable and accurate analytical method for determining these compounds in the crude drug or preparations of the plant. In addition, its volatile chemical composition remains unknown. Objective The main goals were to develop a validated HPLC method for quantifying the active flavonoids (pinostrobin (1), pinocembrin (2) and chrysin (3)) of D. graveolens and to establish its volatile composition. Methodology Separation was carried out on a Licrospher100 RP18 column with a linear gradient acetonitrile 0.1% formic acid and aqueous 0.1% formic acid. Accuracy was determined by spiking the crude drug with the standards, the recoveries were between 99% and 101%. A systematic description of the volatile components of D. graveolens was assessed via GC–MS using headspace solid-phase microextraction (HS–SPME) and hydrodistillation extraction methods. Results The developed HPLC method represented a powerful technique for the quality control of D. graveolens allowing the quantification of the three active flavonoids. For each compound a linear response was evaluated within the range of 0.5–2.0?mg/mL for pinostrobin (1), 0.25–1.25?mg/mL for pinocembrin (2) and 0.05–0.5?mg/mL for chrysin (3). According to SPME the major components in D. graveolens were p-cymene (84.85%) and eucalyptol (11.26%). On the other hand, the essential oil had eucalyptol (42.89%) and p-cymene (16.51%) and did not contain ascaridol. Thus the most relevant volatile components in the species were monoterpenoids. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 5 October 2012 | 10:27 am

HPLC–ESI–QTOF–MS as a Powerful Analytical Tool for Characterising Phenolic Compounds in Olive-leaf Extracts

Introduction Olea europaea L. leaves may be considered a cheap, easily available natural source of phenolic compounds. In a previous study we evaluated the possibility of obtaining bioactive phenolic compounds from olive leaves by pressurised liquid extraction (PLE) for their use as natural anti-oxidants. The alimentary use of these kinds of extract makes comprehensive knowledge of their composition essential. Objective To undertake a comprehensive characterisation of two olive-leaf extracts obtained by PLE using high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC–ESI–QTOF–MS). Method Olive leaves were extracted by PLE using ethanol and water as extraction solvents at 150°C and 200°C respectively. Separation was carried out in a HPLC system equipped with a C18-column working in a gradient elution programme coupled to ESI–QTOF–MS operating in negative ion mode. Results This analytical platform was able to detect 48 compounds and tentatively identify 31 different phenolic compounds in these extracts, including secoiridoids, simple phenols, flavonoids, cinnamic-acid derivatives and benzoic acids. Lucidumoside C was also identified for the first time in olive leaves. Conclusion The coupling of HPLC–ESI–QTOF–MS led to the in-depth characterisation of the olive-leaf extracts on the basis of mass accuracy, true isotopic pattern and tandem mass spectrometry (MS/MS) spectra. We may conclude therefore that this analytical tool is very valuable in the study of phenolic compounds in plant matrices. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 15 September 2012 | 8:41 am

Development, Optimisation and Validation of a Stability-Indicating HPLC Method of Achyrobichalcone Quantification using Experimental Designs

Introduction Achyrobichalcone is a new biflavonoid found in Achyrocline satureioides. It is structurally similar to other bioactive bichalcones that were proven to exert anti-cancer activity. Recently we isolated several achyrobichalcone batches on a semi-preparative scale, showing the need to assess the quality and stability of this substance by analytical methods. Objective To develop and validate a stability-indicating HPLC method of achyrobichalcone quantification using experimental designs. Method The method was developed and optimised by Box–Behnken design using column temperature, flow rate and acetonitrile content in the mobile phase as factors and system suitability parameters as responses. Validation parameters were determined according to official compendiums. Robustness was determined by Plackett–Burman design. Stability of achyrobichalcone was assessed in alkaline, acid, oxidative, thermal and photolytic stress conditions. Results The ideal chromatographic conditions were defined from the optimisation: 37 % of acetonitrile, flow rate of 1.2?mL/min and 33°C temperature. All factors were significant for the resolution between achyrobichalcone and impurities peaks and for the retention factor. The mathematical model developed exhibited a good predictive capacity, and the design proved suitable. The HPLC method was successfully validated, being linear, specific, accurate and precise. The robustness test revealed that the flow rate and detection wavelength should be strictly controlled, as they affect achyrobichalcone concentration. The analyte was unstable only in alkaline media. Conclusion The new method developed affords evaluation of the quality of achyrobichalcone obtained by isolation, and indicates the stability of the molecule under various stress conditions. Copyright © 2012 John Wiley & Sons, Ltd.

Posted on 15 September 2012 | 8:34 am

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