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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:

Application of NIR and MIR spectroscopy for rapid determination of antioxidant activity of Radix Scutellariae from different geographical regions

Introduction The beneficial health effects of traditional Chinese medicines are often attributed to their potent antioxidant activities, usually established in vitro. However, these wet chemical methods for determining antioxidant activities are time-consuming, laborious, and expensive. Objectives This study was conducted to establish a rapid determination of antioxidant activity of Radix Scutellariae using near-infrared (NIR) and mid-infrared (MIR) spectroscopy. Material and methods Antioxidant capabilities were evaluated using 2,2-diphenyl-1-picrylhydrazyl hydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) assays. The total flavonoid contents (TFCs) of Radix Scutellariae were measured by the aluminium chloride colorimetric method. The same sample was then scanned using NIR and MIR spectroscopy. Chemometrics analysis using partial least-squares (PLS) regression was performed to establish the models for predicting the antioxidant activities of Radix Scutellariae. Results A better predictive performance was achieved using PLS models based on NIR data. The determination coefficient (R2) and the residual predictive deviation (RPD) for the validation set were 0.9298 and 2.84 for DPPH, and 0.9436 and 2.66 for TFCs, respectively. MIR-PLS algorithms gave a slightly lower reliability (R2 = 0.9090 and 0.9374, RPD = 2.01 and 2.42, for DPPH and TFC, respectively). Very comparable results for ORAC were obtained with the two methods. Conclusion The developed spectroscopic method can be successfully applied in high-throughput screening of the antioxidant capability of Radix Scutellariae samples. It can also be a viable and advantageous alternative to laborious chemical procedures. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 19 November 2015 | 3:30 am

Screening of peroxynitrite scavengers in Flos Lonicerae by using two new methods, an HPLC-DAD-CL technique and a peroxynitrite spiking test followed by HPLC-DAD analysis

Introduction Peroxynitrite is involved in the pathogenesis of a number of significant diseases. Peroxynitrite scavengers thus have potential application in understanding and treating these diseases. It is, therefore, important to establish screening methods able to rapidly identify peroxynitrite scavengers from herbal plants. Objective To develop effective and easily operable screening methods for identifying peroxynitrite scavengers in complex matrices, including Chinese herbal medicines. Methods Two simple and efficient screening methods have been developed for the identification of natural peroxynitrite scavengers in Flos Lonicerae Japonicae (FLJ). Method I used HPLC-DAD-(luminol-peroxynitrite)-CL techniques combined with Q-TOF MS/MS analysis, while Method II used pre-column reaction of the sample with peroxynitrite, followed by HPLC separation and Q-TOF MS/MS analysis. Results Five peroxynitrite scavengers (neochlorogenic acid, chlorogenic acid, 3,4-O-dicaffeoyl quinic acid, 3,5-O-dicaffeoyl quinic acid and 4,5-O-dicaffeoyl quinic acid) were identified in FLJ using Method I. Besides the compounds identified using Method I, three additional peroxynitrite scavengers (rutin, isoquercitrin and luteoloside) were identified using Method II. Conclusion The two new methods proved to be complementary and the use of these methods should allow rapid detection of peroxynitrite-scavenging natural products from FLJ and other complex matrices. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 16 November 2015 | 10:19 am

Application of comprehensive NMR-based analysis strategy in annotation, isolation and structure elucidation of low molecular weight metabolites of Ricinus communis seeds

Introduction Powder-like extract of Ricinus communis seeds contain a toxic protein, ricin, which has a history of military, criminal and terroristic use. As the detection of ricin in this “terrorist powder” is difficult and time-consuming, related low mass metabolites have been suggested to be useful for screening as biomarkers of ricin. Objective To apply a comprehensive NMR-based analysis strategy for annotation, isolation and structure elucidation of low molecular weight plant metabolites of Ricinus communis seeds. Methodology The seed extract was prepared with a well-known acetone extraction approach. The common metabolites were annotated from seed extract dissolved in acidic solution using 1H NMR spectroscopy with spectrum library comparison and standard addition, whereas unconfirmed metabolites were identified using multi-step off-line HPLC-DAD-NMR approach. Results In addition to the common plant metabolites, two previously unreported compounds, 1,3-digalactoinositol and ricinyl-alanine, were identified with support of MS analyses. Conclusion The applied comprehensive NMR-based analysis strategy provided identification of the prominent low molecular weight metabolites with high confidence. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 14 October 2015 | 10:29 am

Simultaneous Detection of Flavonoids, Phenolic Acids and Alkaloids in Abri Herba and Abri Mollis Herba using Liquid Chromatography Tandem Mass Spectrometry

Introduction Abri Herba has remarkable properties, such as cleanup heat detoxification, dampness and activating blood circulation to dissipate blood stasis; as a result, it has been applied to treat acute or chronic hepatitis and mastitis. Abri mollis Herba is often used as Abri Herba. Hierarchical cluster analysis (HCA) was applied to compare the similarities and differences of the chemical compositions in the two types of medicinal materials. Objective To establish a high-performance liquid chromatography and tandem mass spectrometry (HPLC–MS/MS) method for the simultaneous analysis of 15 flavonoids, two phenolic acids and three alkaloids in Abri Herba and Abri mollis Herba. Methodology The chromatographic separation was performed on a C18 column with a mobile phase of methanol (A), acetonitrile (B) and 0.5‰ acetic acid in water (C) using gradient elution. The detection of the target compounds was performed in multiple-reaction monitoring (MRM) mode using a hybrid quadrupole linear ion trap mass spectrometer equipped with positive/negative ion-switching electrospray ionisation (ESI) source. Results The developed method is reliable, sensitive and specific. In addition, the method has been successfully applied to differentiate 15 batches of Abri Herba and 27 batches of Abri mollis Herba stems. Furthermore, a comparison of the contents among stems, roots and leaves from the same strain in seven batches of Abri mollis Herba and four batches of Abri Herba has also been performed. Conclusion HPLC–MS/MS method is sensitive and selective and can be suitable for the reliable quality control of Abri mollis Herba and Abri Herba. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 28 September 2015 | 9:00 am

Analytical Profiling of Bioactive Phenolic Compounds in Argan (Argania spinosa) Leaves by Combined Microextraction by Packed Sorbent (MEPS) and LC-DAD-MS/MS

Introduction The argan tree (Argania spinosa) is an endemic species from south-western Morocco. Argan-based preparations have been widely used in Moroccan traditional medicine for their biological properties, as well as for several cosmetic purposes. Whereas kernel, pulp of fruit and trunk have been extensively studied for their nutritional and pharmacological effects, relatively little is known about argan tree leaves. Objective The main purpose of the present study is to investigate and characterise the bioactive phenolic fractions in both crude and aqueous extracts derived from argan tree leaves. Methodology A qualitative profile of the antioxidant phenolic compounds in argan leaves was obtained by means of structural hypothesis based on UV spectra and mass spectrometric fragmentation patterns. Moreover, selected phenolics were quantified in argan leaves by using a fully validated method based on liquid chromatography coupled to diode array detection and tandem mass spectrometry (LC-DAD-MS/MS). All the extracts were purified by a fast and reliable microextraction by packed sorbent (MEPS) procedure, before analysing them by LC-MS/MS. Results Based on retention times, mass spectrometric fragmentation and UV spectra, 13 phenolic compounds were identified or tentatively elucidated from crude and aqueous extracts derived from Argania spinosa leaves, while seven compounds were quantified in both extracts. Conclusion The obtained results could represent a first step towards a complete characterisation of the argan plant, its bioactive profiling and the valorisation of its by-products as a source of potentially beneficial bioactive molecules. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 9 September 2015 | 6:30 pm

Quantitation of phenylpropanoids and iridoids in insulin-sensitising extracts of Leonurus sibiricus L. (Lamiaceae)

Introduction Leonurus sibiricus L. is regularly used in traditional Mongolian medicine including for the treatment of symptoms of diabetes mellitus. Objectives To provide a validated quantitation method for the quality control of Leonurus sibiricus and to prove in vitro insulin-sensitisation, thereby supporting the traditional use of Leonurus sibiricus. Methodology Pulverised Leonurus sibiricus material was either extracted with methanol or methanol:water (25:75, v/v). HPLC-CAD (charged aerosol detector) separations were performed on a Luna Phenyl-Hexyl column with water and acetonitrile (both modified with 0.1% formic acid) as mobile phase. Gradient elution was employed using theophylline as internal standard. Tentative peak identification was facilitated by HPLC-MS. Validation was carried out according to ICH (International Conference on Harmonisation) guidelines. Potential insulin-sensitisation of accordant extracts was assessed in glucose uptake experiments in C2C12 myocytes and protein tyrosine phosphatase 1B (PTP1B) enzyme assays. Results Thirty-six compounds were tentatively identified based on their retention times, UV spectra, MS fragments and data from literature. They comprise phenolcarboxylic acids, flavonoids, iridoid glycosides, and phenylpropanoids, among which acetylharpagide, ajugoside, lavandulifolioside, and verbascoside were selected for quantitation. The methanol extract contained 0.42% combined iridoids, and 1.58% combined phenylpropanoids. Validation showed good accuracy, intermediate precision and robustness. The methanol extract of Leonurus sibiricus led to a 1.5 fold increase in insulin-stimulated cellular glucose uptake and inhibition of PTP1B by 40% at a concentration of 10 µg/mL. Conclusion HPLC-CAD analysis allowed sensitive quantitation of the selected marker compounds in Leonurus sibiricus, thereby providing a reliable tool for its quality control. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 2 September 2015 | 9:11 am

Use of time-resolved spectroscopy as a method to monitor carotenoids present in tomato extract obtained using ultrasound treatment

Introduction Compounds exhibiting antioxidant activity have received much interest in the food industry because of their potential health benefits. Carotenoids such as lycopene, which in the human diet mainly derives from tomatoes (Solanum lycopersicum), have attracted much attention in this aspect and the study of their extraction, processing and storage procedures is of importance. Optical techniques potentially offer advantageous non-invasive and specific methods to monitor them. Objectives To obtain both fluorescence and Raman information to ascertain if ultrasound assisted extraction from tomato pulp has a detrimental effect on lycopene. Method Use of time-resolved fluorescence spectroscopy to monitor carotenoids in a hexane extract obtained from tomato pulp with application of ultrasound treatment (583 kHz). The resultant spectra were a combination of scattering and fluorescence. Because of their different timescales, decay associated spectra could be used to separate fluorescence and Raman information. This simultaneous acquisition of two complementary techniques was coupled with a very high time-resolution fluorescence lifetime measurement of the lycopene. Results Spectroscopic data showed the presence of phytofluene and chlorophyll in addition to lycopene in the tomato extract. The time-resolved spectral measurement containing both fluorescence and Raman data, coupled with high resolution time-resolved measurements, where a lifetime of ~5 ps was attributed to lycopene, indicated lycopene appeared unaltered by ultrasound treatment. Detrimental changes were, however, observed in both chlorophyll and phytofluene contributions. Conclusion Extracted lycopene appeared unaffected by ultrasound treatment, while other constituents (chlorophyll and phytofluene) were degraded. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 20 August 2015 | 11:20 am

Simultaneous quantitative determination of multiple bioactive markers in Ocimum sanctum obtained from different locations and its marketed herbal formulations using UPLC-ESI-MS/MS combined with principal component analysis

Introduction Ocimum sanctum L., with phenolic acids, flavonoids, propenyl phenols and terpenoids as active pharmacological constituents, is a popular medicinal herb and is present as an ingredient in many herbal formulations. Therefore, development of a reliable analytical method for simultaneous determination of the pharmacologically active constituents of O. sanctum is of high importance. Objective To develop and validate a new, rapid, sensitive and selective UPLC–ESI/MS/MS method for simultaneous determination of 23 bioactive markers including phenolic acids, flavonoids, propenyl phenol and terpenoid in the leaf extract and marketed herbal formulations of O. sanctum. Methods An UPLC–ESI/MS/MS method using negative electrospray ionisation (ESI) in multiple-reaction-monitoring (MRM) mode was used for simultaneous determination. Chromatographic separation was achieved on an Acquity UPLC BEH C18-column using a gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Principal component analysis (PCA) was applied to correlate and discriminate eight geographical collections of O. sanctum based on quantitative data of the analytes. Results The developed method was validated as per International Conference on Harmonization guidelines and found to be accurate, with overall recovery in the range 95.09–104.84% (RSD???1.85%), precise (RSD???1.98%) and linear (r2???0.9971) over the concentration range of 0.5–1000?ng/mL. Ursolic acid was found to be the most abundant marker in all the samples investigated, except for the marketed tablet. Conclusion The method established is simple, rapid and sensitive, hence it can be reliably utilised for the quality control of O. sanctum and derived herbal formulations. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 13 August 2015 | 8:23 am

Multi-response optimisation of ultrasound-assisted extraction for recovery of flavonoids from red grape skins using response surface methodology

Introduction For the characterisation of grape cultivars, the profile and content of flavonoids are important because these compounds impact grape and wine quality. To determine the correct profile and content of flavonoids, the use of robust, sensitive and reliable methods is necessary. Objective The object of this research is to develop a new ultrasound-assisted extraction (UAE) method for the recovery of flavonoids from grape skins using response surface methodology. Method Optimisation of UAE was performed using a complementary study combining a Box-Behnken experimental design with qualitative analysis by high-performance liquid chromatography. Results Optimal extraction conditions were obtained using the extraction solvent composed of acetonitrile:water:formic acid (26:73:1, v/v/v) at an extraction temperature of 50°C, an extraction time of 15?min in a single-extraction step and with a solid-to-solvent ratio of 1:80?g/mL. The calculated relative standard deviations for the optimal extraction method were very low, measuring less than 5%. Conclusions This study demonstrates that numerous factors have strong effects on the extraction efficiency, including the type of organic modifier and its percentage in the extraction solvent, the number of extraction steps, the solid-to-solvent ratio, the extraction time and temperature and, finally, the particular nature of analyte and their position within the grape skin cell. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 6 August 2015 | 7:18 pm

A new TLC bioautographic assay for qualitative and quantitative estimation of lipase inhibitors

Introduction Lipase inhibitory assays based on TLC bioautography have made recent progress; however, an assay with greater substrate specificity and quantitative capabilities would advance the efficacy of this particular bioassay. Objective To address these limitations, a new TLC bioautographic assay for detecting lipase inhibitors was developed and validated in this study. Methods The new TLC bioautographic assay was based on reaction of lipase with ?-naphthyl myristate and the subsequent formation of the purple dye between ?-naphthol and Fast Blue B salt (FBB). The relative lipase inhibitory capacity (RLIC) was determined by a TLC densitometry with fluorescence detection, expressed as orlistat equivalents in millimoles on a per sample weight basis. Six pure compounds and three natural extracts were evaluated for their potential lipase inhibitory activities by this TLC bioautographic assay. Results The ?-naphthyl myristate as the substrate improved the detection sensitivity and specificity significantly. The limit of detection (LOD) of this assay was 0.01?ng for orlistat, the current treatment for obesity. This assay has acceptable accuracy (92.07–105.39%), intra-day and inter-day precisions [relative standard deviation (RSD), 2.64–4.40%], as well as intra-plate and inter-plate precisions (RSD, 1.8–4.9%). Conclusion The developed method is rapid, simple, stable, and specific for screening and estimation of the potential lipase inhibitors. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 29 July 2015 | 12:22 pm

Seasonal Variation of Triterpenes and Phenolic Compounds in Australian Centella asiatica (L.) Urb

Introduction Specific triterpenes, phenolic acids and flavonoids in Centella asiatica have been found to be bioactive. Harvesting the plant when these putative bioactive compounds are at their highest concentrations would provide consistency in their chemical profile, thus ensuring the quality and efficacy of derived medicinal products. Objective The aim of the study was to determine the impact of harvesting time on the contents of major triterpenoid and phenolic compounds in C. asiatica. Methodology Australian C. asiatica was collected from a designated area in different months. The principal triterpenes (asiaticoside, madecassoside, asiatic acid and madecassic acid), flavonoid compounds (rutin, quercetin and kaempferol) and chlorogenic acid were quantitatively determined by HPLC-DAD analysis. Results Triterpenoid, kaempferol and chlorogenic acid content showed significant variation (p?<?0.05) in different collecting months. The total content of the four triterpenes reached its highest levels in January and February (83.15?±?0.16?mg/g and 78.41?±?0.16?mg/g, respectively), the summer season of the southern hemisphere, and their lowest values in winter (June) and spring (October) seasons (35.65?±?0.20 and 35.50?±?0.55?mg/g, respectively). Similarly, the contents of chlorogenic acid and kaempferol were the highest in December and January (1.62?±?0.01 and 0.33?±?0.01?mg/g, respectively), and the lowest in June (0.06?±?0.01 and 0.09?±?0.01?mg/g, respectively). Conclusion The results indicate that harvesting C. asiatica in summer returns the highest yield of the target triterpenoids, kaempferol and chlorogenic acid. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 29 July 2015 | 12:21 pm

Sequential injection analysis with chemiluminescence detection for rapid monitoring of commercial Calendula officinalis extractions

Introduction Plant extracts containing high levels of antioxidants are desirable due to their reported health benefits. Most techniques capable of determining the antioxidant activity of plant extracts are unsuitable for rapid at-line analysis as they require extensive sample preparation and/or long analysis times. Therefore, analytical techniques capable of real-time or pseudo real-time at-line monitoring of plant extractions, and determination of extraction endpoints, would be useful to manufacturers of antioxidant-rich plant extracts. Objectives To develop a reliable method for the rapid at-line extraction monitoring of antioxidants in plant extracts. Materials and Methods Calendula officinalis extracts were prepared from dried flowers and analysed for antioxidant activity using sequential injection analysis (SIA) with chemiluminescence (CL) detection. The intensity of CL emission from the reaction of acidic potassium permanganate with antioxidants within the extract was used as the analytical signal. The SIA-CL method was applied to monitor the extraction of C. officinalis over the course of a batch extraction to determine the extraction endpoint. Results were compared with those from ultra high performance liquid chromatography (UHPLC). Results Pseudo real-time, at-line monitoring showed the level of antioxidants in a batch extract of Calendula officinalis plateaued after 100 min of extraction. These results correlated well with those of an offline UHPLC study. Conclusion SIA-CL was found to be a suitable method for pseudo real-time monitoring of plant extractions and determination of extraction endpoints with respect to antioxidant concentrations. The method was applied at-line in the manufacturing industry. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 29 July 2015 | 12:21 pm

A Novel Four-step Approach for Systematic Identification of Naphthoquinones in Juglans cathayensis Dode using Various Scan Functions of Liquid Chromatography-Tandem Mass Spectrometry along with Data Mining Strategies

Introduction Systematic analyses of naphthoquinones in Juglans cathayensis have not yet been reported. It is very challenging to identify naphthoquinones with various structural diversities, especially those at trace levels. Objective To develop an efficient analytical approach for systematic discovery and identification of naphthoquinones in Juglans cathayensis. Methodology A novel four-step approach was evaluated by utilizing various scan functions of liquid chromatography-triple quadrupole-linear ion trap mass spectrometry (LC-QTRAP-MS/MS) and liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) along with data mining strategies. First, MS/MS fragmentation behaviors of naphthoquinones were investigated. Second, multiple ion monitoring triggered enhanced product ion scan (MIM-EPI) with specified ions was conducted to identify targeted naphthoquinones. Third, other scan functions of QTRAP-MS/MS and data mining strategies were explored to identify untargeted naphthoquinones. Fourth, structural rationalization and confirmation of naphthoquinones were performed using QTOF-MS/MS via its accurate mass measurement and MS/MS fragmentation functions. Results Optimal scan methods and data mining strategies using QTRAP-MS/MS were obtained for identification of targeted and untargeted naphthoquinones. Consequently, 48 naphthoquinones including 24 novel ones were identified or tentatively identified from Juglans cathayensis. Conclusion A novel four-step approach for efficient discovery and identification of naphthoquinones was developed by exploring various scan functions of current LC-MS/MS technologies and data mining strategies, providing an example for systematic characterization of certain classes of phytochemicals, especially trace analytes in complex samples. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 20 July 2015 | 10:37 am

Rapid identification of new minor chemical constituents from Smilacis Glabrae Rhizoma by combined use of UHPLC-Q-TOF-MS, preparative HPLC and UHPLC-SPE-NMR-MS techniques

Introduction Herbs are an important resource for new drug development. However, the conventional approach for the discovery of new compounds from herbs was time-consuming, tedious, and inefficient. Objectives Establish a quick approach to identify new minor constituents in herbs. Methods The constituents in herbs were firstly analysed using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Based on the accurate masses, isotopic ions, and the characteristic fragmentation ions in the mass spectra, the molecular compositions and possible structures of compounds were first deduced. After being enriched by a preparative HPLC method, the potential new minor structures were definitely identified by an on-line UHPLC-solid phase extraction-nuclear magnetic resonance-mass spectrometry (UHPLC-SPE-NMR-MS) approach. Results By combined the use of UHPLC-Q-TOF-MS, preparative HPLC and UHPLC-SPE-NMR, three new minor compounds were definitely identified as bis-3,4-dihydroxyphenylpropanoid-substituted catechins (A2 and A3) and 4?-formyl-astilbin (B5). In addition, five isomers of bis-dihydroxyphenylpropanoid-substituted catechin (A1, A4–A7), four isomers of 4?-formyl-astilbin (B1–B4), engeletin formates and isomers (C1–C5), formyl-cinchonains (D1–D4), formyl-caffeoylshikimic acid (E1–E4) were also tentatively determined by MS and MS/MS characterisation. Conclusion The combination of UHPLC-Q-TOF-MS, preparative HPLC and UHPLC-SPE-NMR-MS techniques is a quick and effective approach for finding new minor constitutes from herbs. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 17 July 2015 | 8:35 am

Thin Layer Chromatography-Autography-High Resolution Mass Spectrometry Analysis: Accelerating the Identification of Acetylcholinesterase Inhibitors

Introduction The prevailing treatment for Alzheimer's disease is the use of acetylcholinesterase (AChE) inhibitors. Natural extracts are the principal source of AChE's inhibitors. However, their chemical complexity demands for simple, selective and rapid assays. Objective To develop a strategy for identification of AChE inhibitors present in mixtures employing high resolution mass spectrometry (HRMS) and thin layer chromatography (TLC)-biological staining. Methodology The strategy uses an autographic assay based on the ?-naphthyl acetate – fast blue B system for the detection of AChE activity. The immobilisation of AChE in agar allowed the extraction of the compounds for analysis by HRMS. Three TLC experiments employing different solvent systems were used in parallel and the mass spectra of the compounds extracted from the inhibition halos, were compared. The analysis was performed under MatLab environment. Results The strategy was used to detect the presence of physostigmine in an extract of Brassica rapa L. spiked with the inhibitor. Similarly, caffeine was straightforwardly spotted as responsible for the inhibitory properties of an extract of Ilex paraguariensis Saint-Hilaire. Comparison of the HRMS profiles lead to the facile identification of the [M+H]+ and [M+Na]+ of the compounds responsible for the inhibition. Conclusion The proposed methodology, coupling TLC-AChE autography-HRMS, illustrates the feasibility of assigning molecular formulas of active compounds present in complex mixtures directly from autography. The new AChE agar-immobilised assay presented a more homogenous colour and a better definition than direct spraying methods, reducing the cost of the assay and improving its sensitivity. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 23 June 2015 | 1:24 pm

Rapid Separation of Three Proanthocyanidin Dimers from Iris lactea Pall. var. Chinensis (Fisch.) Koidz by High-Speed Counter-Current Chromatography With Continuous Sample Load and Double-Pump Balancing Mode

Introduction The dried seeds of Iris lactea have been used in traditional Chinese medicine. Previous studies have been focused on irisquinones while other chemical components are rarely reported. Objective To establish an efficient high-speed counter-current chromatography (HSCCC) separation method with continuous sample load (CSL) and double-pump balancing (DPB) mode to isolate proanthocyanidins from I. lactea. Methods Firstly, an ethyl acetate extract of I. lactea was pre-fractionated by silica column chromatography for the enrichment of proanthocyanidins. Secondly, the enriched proanthocyanidins sample (EPS) was further fractionated by HSCCC with a two-phase solvent system ethyl acetate:n-butanol:water (9:1:10, v/v/v) using DPB mode. The flow rate of the two phases was 2.2?mL/min, the revolution speed was 900?rpm, the separation temperature was 30?°C and the detection wavelength was 280?nm. Finally, the structures of the three isolated proanthocyanidins were elucidated by spectroscopic methods and compared with published data. Results Under the optimized conditions, 600?mg of the EPS with six continuous injections (100?mg/time) was fractionated, yielding 57?mg of prodelphinidin B3, 198?mg of procyanidin B3, and 162?mg of procyanidin B1, at purities of 97.2%, 98.1% and 97.3%, respectively. Conclusions The HSCCC separation method with CSL and DPB proved to be rapid, convenient and economical, constituting an efficient strategy for the isolation of proanthocyanidins. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 23 June 2015 | 1:23 pm

In-field Volatile Analysis Employing a Hand-held Portable GC-MS: Emission Profiles Differentiate Damaged and Undamaged Yellow Starthistle Flower Heads

Introduction Understanding the complex chemical signalling of plants and insects is an important component of chemical ecology. Accordingly, the collection and analysis of chemical cues from plants in their natural environment is integral to elucidation of plant–insect communications. Remote plant locations and the need for a large number of replicates make in situ headspace analyses a daunting logistical challenge. A hand-held, portable GC-MS system was used to discriminate between damaged and undamaged Centaurea solstitialis (yellow starthistle) flower heads in both a potted-plant and natural setting. Objective To determine if a portable GC-MS system was capable of distinguishing between undamaged and mechanically damaged plant treatments, and plant environments. Methodology A portable GC-MS utilising needle trap adsorbent technology was used to collect and analyse in situ headspace volatiles of varying yellow starthistle treatments. Principal component analysis (PCA) was used to distinguish treatments and identify biomarker volatiles. Analysis of variance (ANOVA) was used to determine differences between treatment volatile amounts. Results The portable GC-MS system detected 31 volatiles from the four treatments. Each GC-MS run was completed in less than 3?min. PCA showed four distinct clusters representing the four treatments – damaged and undamaged potted plant, and damaged and undamaged natural plant. Damage-specific volatiles were identified. Conclusion The portable GC-MS system distinguished the treatments based on their detected volatile profiles. Additional statistical analysis identified five possible biomarker volatiles for the treatments, among them cyclosativene and copaene, which indicated damaged flower heads. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 11 June 2015 | 5:55 pm

Development of a Rapid Immunochromatographic Strip Test for the Detection of Mulberroside A

Introduction Mulberroside A (MuA) is the major active anti-tyrosinase compound in the root bark extract of Morus alba L. (Moraceae). Typically, MuA is widely employed as an active ingredient in whitening cosmetics. A rapid and simple assay system utilizing a small quantity of test sample is essential for the detection of MuA in large number of samples. An immunoassay using highly specific MuA polyclonal antibodies may be useful for the determination of small quantities of MuA in test samples. Objective To establish a rapid qualitative MuA test, an immunochromatographic strip test was developed using anti-MuA polyclonal antibodies (anti-MuA PAb). Methodology The qualitative assay was based on a competitive immunoassay where the detection reagent consisted of anti-MuA PAb colored with colloidal gold particles. The capture reagent was a MuA-ovalbumin (MuA-OVA) conjugate immobilized on the test strip membrane. Results A sample containing MuA and the detection reagent were incubated together with immobilized capture reagent on a nitrocellulose membrane. When MuA was present, it competed with the immobilized conjugates on the strip membrane to bind a limited amount of colored antibodies; thus, a positive sample showed no color on the capture spot zone. The detection limit for the strip test was 2?µg/mL. The developed immunochromatographic strip test was utilized to determine MuA in plants, medical preparations and cosmetic samples. Conclusion This immunochromatographic strip test is advantageous as a rapid, simple and sensitive screening method for the detection of MuA in plant extracts, cosmetic samples and pharmaceutical products.

Posted on 11 June 2015 | 5:53 pm

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