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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:

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

Direct Infusion ESI-IT-MSn Alkaloid Profile and Isolation of Tetrahydroharman and Other Alkaloids from Bocageopsis pleiosperma Maas (Annonaceae)

Introduction The Annonaceae family is known as a promising abundant source of secondary metabolites, especially annonaceous acetogenins, terpenoids and isoquinoline-derived alkaloids. Although widely investigated from the phytochemical viewpoint, this family still presents some largely unexplored genera, e.g. the Bocageopsis. Objective To investigate the alkaloid content of Bocageopsis pleiosperma Maas using direct infusion electrospray ionisation ion trap tandem mass spectrometry (ESI-IT-MSn) analysis. Methodology Dichloromethane extracts of aerial parts were subjected to acid–base partitioning to yield the alkaloidal fractions. These fractions were analysed by direct infusion into a (+)ESI-IT-MSn system. The alkaloidal fraction from the leaves was also obtained on a large scale and subjected to chromatographic separation. Results The tentative MSn-based identification of alkaloids in leaves, twigs and trunk bark showed that aporphine alkaloids were restricted to the leaves and twigs, tetrahydroprotoberberine alkaloids were only found in the twigs and trunk bark while benzylisoquinoline alkaloids were found in the leaves, twigs and trunk bark. Chromatographic separation of the leaf alkaloidal fraction yielded the aporphine alkaloids nornuciferine, asimilobine and isoboldine, the ?-carboline alkaloid tetrahydroharman and some mixtures containing benzylisoquinoline and aporphine alkaloids, all described for the first time in the Bocageopsis genus. Furthermore, tetrahydroharman has not previously been reported in the Magnoliales order. Conclusion Direct infusion ESI-IT-MSn analysis of alkaloids allowed fast recognition of alkaloidal classes previously reported in the Annonaceae family, aiding the chromatographic step and allowing a selective isolation of compounds previously not identified in the Bocageopsis genus. Copyright © 2015 John Wiley & Sons, Ltd. Alkaloid content of Bocageopsis pleiosperma was investigated by direct infusion ESI-IT-MSn analysis. Chromatographic separation yielded aporphine, ?-carboline and some mixtures containing benzylisoquinoline and aporphine alkaloids. All alkaloids were described for the first time in the Bocageopsis genus, being tetrahydroharman described for the first time in Magnoliales order.

Posted on 24 June 2015 | 2:23 pm

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

Determination of Terpenoid Indole Alkaloids in Hairy Roots of Rhazya stricta (Apocynaceae) by GC-MS

Introduction Rhazya stricta Decne. (Apocynaceae) is a medicinal plant rich in terpenoid indole alkaloids (TIAs), some of which possess important pharmacological properties. The study material including transgenic hairy root cultures have been developed and their potential for alkaloid production are being investigated. Objective In this study, a comprehensive GC-MS method for qualitative and quantitative analysis of alkaloids from Rhazya hairy roots was developed. Methods The composition of alkaloids was determined by using GC-MS. In quantification, the ratio between alkaloid and internal standard was based on extracted ion from total ion current (TIC) analyses. Results The developed method was validated. An acceptable precision with RSD???8% over a linear range of 1 to 100?µg/mL was achieved. The accuracy of the method was within 94–107%. Analysis of hairy root extracts indicated the occurrence of a total of 20 TIAs. Six of them, pleiocarpamine, fluorocarpamine, vincamine, ajmalicine and two yohimbine isomers are reported here for the first time in Rhazya. Trimethylsilyl (TMS) derivatisation of the extracts resulted in the separation of two isomers for yohimbine and also for vallesiachotamine. Clearly improved chromatographic profiles of TMS-derivatives were observed for vincanine and for minor compounds vincamine and rhazine. Conclusion The results show that the present GC-MS method is reliable and well applicable for studying the variation of indole alkaloids in Rhazya samples. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 11 June 2015 | 5:54 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

Hollow Fibre Liquid Phase Micro-extraction by Facilitated Anionic Exchange for the Determination of Flavonoids in Faba Beans (Vicia faba L.)

Introduction Flavonoids are polyphenolic compounds found ubiquitously in foods of plant origin. They are commonly extracted from plant materials with ethanol, methanol, water, their combination or even with acidified extracting solutions. The disadvantages of these methods are the use of high quantity of organic solvent, the possible loss of analytes in the different steps and the laborious process of the techniques. In addition, the complexity of the phenolic mixtures present in plant materials requires a preliminary clean-up and fractionation of the crude extracts. Objective To develop a hollow fibre liquid phase micro-extraction (HF-LPME) method for a one step clean-up and pre-concentration of flavonoids. Methodology Two flavonoids (catechin and rutin) has been extracted by HF-LPME and analysed by HPLC. The related driving force for the liquid membrane has been studied by means of facilitated and non-facilitated transport. Different ionic and non-ionic water insoluble compounds [trioctylamine (TOA), tributyl phosphate (TBP), trioctylphosphine oxide (TOPO) and methyltrioctylammonium chloride (aliquat 336)] were used as carriers. The liquid membrane was constituted by a solution of n-decanol in the presence or absence of carriers. Results Maximum enrichment factors were obtained with n-decanol/aliquat 336 (20%) as organic liquid membrane, sodium hydroxide (NaOH) (0.1?M) as donor solution, sodium chloride (NaCl) (2?M) as acceptor solution and 3?h as extraction time. Under these conditions, good results for validation parameters were obtained [for linearity, limit of detection (LOD), limit of quantitation (LOQ) and repeatability]. Conclusions The developed method is simple, effective and has been successfully applied to determine catechin and rutin in ethanolic extracts of faba beans. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 5 June 2015 | 1:58 pm

Cluster Analysis of Commercial Samples of Bauhinia spp. Using HPLC-UV/PDA and MCR-ALS/PCA Without Peak Alignment Procedure

Introduction Bauhinia forficata Link. is recognised by the Brazilian Health Ministry as a treatment of hypoglycemia and diabetes. Analytical methods are useful to assess the plant identity due the similarities found in plants from Bauhinia spp. HPLC-UV/PDA in combination with chemometric tools is an alternative widely used and suitable for authentication of plant material, however, the shifts of retention times for similar compounds in different samples is a problem. Objective To perform comparisons between the authentic medicinal plant (Bauhinia forficata Link.) and samples commercially available in drugstores claiming to be “Bauhinia spp. to treat diabetes” and to evaluate the performance of multivariate curve resolution – alternating least squares (MCR-ALS) associated to principal component analysis (PCA) when compared to pure PCA. Methodology HPLC-UV/PDA data obtained from extracts of leaves were evaluated employing a combination of MCR-ALS and PCA, which allowed the use of the full chromatographic and spectrometric information without the need of peak alignment procedures. Results The use of MCR-ALS/PCA showed better results than the conventional PCA using only one wavelength. Only two of nine commercial samples presented characteristics similar to the authentic Bauhinia forficata spp., considering the full HPLC-UV/PDA data. Conclusion The combination of MCR-ALS and PCA is very useful when applied to a group of samples where a general alignment procedure could not be applied due to the different chromatographic profiles. This work also demonstrates the need of more strict control from the health authorities regarding herbal products available on the market. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 5 June 2015 | 1:58 pm

Quantification of Tannins and Related Polyphenols in Commercial Products of Tormentil (Potentilla tormentilla)

Introduction Potentilla tormentilla has many biological and pharmacological properties and can be used as an ingredient of some herbal medicines or beverages. Objective The aim of this study was to evaluate the content of individual polyphenols, especially condensed and hydrolysable tannins in commercially available tormentil rhizomes and tinctures using chromatographic methods. Methods A quantitative analysis (HPLC-PDA) was preceded by qualitative studies (UPLC-qTOF-MS/MS) and the isolation (CC) of the major tannin compounds. Results The tested plant material is characterised by a high content of tannins and related polyphenols, i.e. in rhizomes even at the level above 20% and in tinctures above 2%. The main components of tormentil rhizomes are procyanidin B3 (mean?~?3.6%), procyanidin C2 (mean?~?2.8%), agrimoniin (mean?~?2.5%), 3-O-galloylquinic acid (mean?~?1.7%), catechin (mean?~?1.6%), other flavan-3-ol oligomers (mean?~?0.5–1.1) and laevigatins (mean?~?0.2–0.6%). Free ellagic acid and glycosides of ellagic and methylellagic acids are secondary components. Conclusions Underground parts of tormentil are a source of oligomeric proanthocyanidins and ellagitannins, but in smaller quantity of gallotannins. Monogalloylquinic acids are new identified compounds, which had not been described in Potentilla tormentilla before we started our research. In the analysed tormentil tinctures agrimoniin concentration is lower in relation to other tannins. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 5 June 2015 | 1:57 pm

Quantitative and Qualitative Investigations of Pharmacopoeial Plant Material Polygoni Avicularis Herba by UHPLC-CAD and UHPLC-ESI-MS Methods

Introduction Polygonum aviculare L. also known as common knotgrass is an annual herbaceous weed occurring all over the world in the temperate regions. Recent studies report that flavonol glucuronides are major constituents of common knotgrass. There is no comprehensive analytical procedure for the standardisation of Polygoni Avicularis Herba available on the European market. Objective To develop a method for the proper authentication and standardisation of Polygoni Avicularis Herba and to preliminary evaluate variability in qualitative and quantitative composition among commercial samples and samples from wild harvesting defined as Polygonum aviculare sensu lato. Methodology The UHPLC-ESI(+)-MS method was used for the qualitative screening of nine independent samples of Polygonum aviculare herb. The UHPLC-CAD method was developed for the quantitation of the major compounds in an extract using quercetin-3-O-glucuronide as a standard. Results Twenty-five major constituents were detected and characterised. Among them three new natural products were tentatively identified. Twelve compounds were quantitated using a validated UHPLC-CAD method. In all nine samples flavonol glucuronides were confirmed as major compounds. The total flavonoid content was estimated for all samples and varied from 0.70 to 2.20%. Conclusion The developed procedure may be used for the routine standardisation of common knotgrass. The results indicate that the pharmacopoeial approach to the authentication and standardisation of Polygonum aviculare herb should be revised. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 5 June 2015 | 1:54 pm

Arylnaphthalene and Aryltetralin-type Lignans in Hairy Root Cultures of Linum perenne, and the Stereochemistry of 6-Methoxypodophyllotoxin and One Diastereoisomer by HPLC-MS and NMR Spectroscopy

Introduction Hairy root cultures of Linum sp. are an alternative for the high production of lignans. Linum perenne is known to produce arylnaphthalene-type lignans such as justicidin B, isojusticidin and diphyllin. Objective To elucidate the presence of aryltetralin-type lignan diastereoisomers, besides the known arylnaphthalene-type lignans, in hairy roots of Linum perenne, and to determine the configurations of one diastereoisomer of 6-methoxypodophyllotoxin (6-MPTOX). Methods Lignans from hairy root cultures of Linum perenne were extracted and separated by HPLC. Arylnaphthalene-type lignans were identified by LC-MS, according to the literature. Two diastereoisomers of aryltetralin-type lignans were analysed by mass spectrometry and NMR spectroscopy. Results Numerous arylnaphthalene-type lignans (diphyllin-2-hexose-pentose, diphyllin-3-pentose and diphyllin-hexose) were identified in hairy root cultures. Methoxypodophyllotoxin, an aryltetralin-type lignan, was also identified, as well as one diastereoisomer. This aryltetralin-type lignan could be derived via 7-hydroxymatairesinol as a hypothetical biosynthetic pathway. The stereochemical configurations of aryltetralin isomers were determined. Conclusion Arylnaphthalene and two diastereoisomers of aryltetralin-type lignans are produced in Linum perenne hairy root cultures. Matairesinol, the precursor of justicidin B, also seems to be converted into 6-MPTOX via 7-hydroxymatairesinol. This is the first report of the stereochemical configurations of an aryltetralin-type lignan other than podophyllotoxin (PTOX). Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 15 May 2015 | 4:05 pm

LC-MS-based Metabolite Profiling of Methanolic Extracts from the Medicinal and Aromatic Species Mentha pulegium and Origanum majorana

Introduction There has been increasing interest dedicated to the phenolic compounds with a view to their antioxidant and healthy properties. Recent studies have focused on plants from the Lamiaceae family with special interest in phenolic compounds antioxidant potential. Objective The metabolite profile of methanolic extracts from two Lamiacea medicinal plants was investigated. Materials and Methods Mentha pulegium and Origanum majorana methanolic extracts were analysed using reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled to electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) detection in the negative ion mode. Results A total of 85 metabolites were characterised from different families, such as organic acids and derivatives, amino acids and derivatives, nucleosides, phenolic compounds as well as other polar metabolites, by using the MS and MS/MS information provided by the QTOF-MS. However, the total phenols and flavonoids were also quantified spectrophotometrically and they registered higher amounts in Mentha pulegium than in Origanum majorana extract. Gallocatechin was the major compound in M. pulegium extract whereas quercetin dimethyl ether, jaceidin and dihydrokaempferide were the major ones in O. majorana extract. Conclusion The distribution of phenolic compounds in the methanolic extract showed a variation among studied plants. Mentha pulegium can be considered as a source of gallocatechin. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 15 May 2015 | 4:04 pm

The Minor Ecdysteroids from Ajuga turkestanica

Introduction Ajuga turkestanica is a plant used in traditional medicine for its high ecdysteroid content, including the presence of the particularly active turkesterone, which possess efficient anabolic activity. Objectives To isolate and identify minor ecdysteroids present in a semi-purified plant fraction containing ca. 70% turkesterone. Material and Methods Multi-step preparative HPLC (combining RP- and NP-HPLC systems) was used to purify the different components present in the turkesterone fraction. Isolated compounds were identified by high-resolution mass spectrometry and 2D-NMR. Results Fourteen ecdysteroids (including turkesterone and 20-hydroxyecdysone) were isolated. Seven of these, all bearing an 11?-hydroxy group, were previously unreported. Conclusion Ajuga turkestanica ecdysteroids are characterised by the abundance of 11?-hydroxylated compounds and by the simultaneous presence of 24C, 27C, 28C and 29C ecdysteroids. It is expected that even more ecdysteroids are to be found in this plant since the starting material for this study lacked the less polar ecdysteroids. The simultaneous presence of 20-hydroxyecdysone and turkesterone (its 11?-hydroxy analogue) as the two major ecdysteroids suggests that every ecdysteroid is probably present in both 11?-hydroxy and 11-deoxy forms. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 8 May 2015 | 9:48 am

Efficient Combination of Circulating Ultrasound-assisted Extraction and Centrifugal Partition Chromatography for Extraction and On-line Separation of Chemical Constituents from Stellera chamaejasme L.

Introduction Sample preparation is a crucial step in medicinal herb analysis because the desired chemical components need to be extracted from the herbal materials for further separation and characterisation. Thus, the development of " modern" sample preparation techniques with significant advantages over conventional methods is very important. Objective The aim of this study was the development of a new preparation method using circulating ultrasonic-assisted extraction (CUAE) coupled with centrifugal partition chromatography (CPC) for continuous extraction and on-line isolation of chemical constituents from Stellera chamaejasme L. Methodology The stationary or mobile phase was used as the extraction solvent. Extraction parameters, including the ultrasound power, extraction time, temperature, and liquid:solid ratio, were optimised using a response surface methodology. Results The extraction time, temperature, and power considerably affected the extraction yield. The optimised extraction parameters were an ultrasound power of 800?W, extraction time of 30?min, extraction temperature of 70?°C, and liquid:solid ratio of 8?mL/g. The solvent system for CUAE and CPC was optimised using mathematical equations, and the two-phase solvent system of n-hexane:ethyl acetate:methanol:water at a volume ratio of 3:5:4:6 was calculated. Four target compounds (daphnoretin, chamaechromone, neochamaejasmin A, and isochamaejasmin) with purities above 96% were successfully extracted and isolated on-line via CUAE/CPC. Conclusion Compared with the reference extraction methods, the instrumental setup achieved a scientific and systematic extraction and isolation of natural products and has great potential for industrial application. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 22 April 2015 | 9:22 am

Multiresponse Optimisation Applied to the Development of a TLC Autography for the Detection of Tyrosinase Inhibitors

Introduction Autographic methods are useful tools to detect bioactive compounds in complex matrixes. Experimental design and optimisation techniques were implemented for the development of an autographic assay suitable for the detection of tyrosinase inhibitors. Objectives To develop an autographic assay to detect tyrosinase inhibitors using gel entrapped enzyme, experimental design and response surface methodology (RSM) to optimise conditions with a minimum number of experiments. Methods Gel entrapment was used for the assay and the effects of four factors on the sensitivity and the detection limit for known inhibitors of the enzyme were evaluated. The factors were: tyrosinase amount (TA), L-tyrosine amount (LTA), incubation time and incubation temperature. Results The assay allowed the detection of kojic acid in an extract of Calamagrostis viridiflavescens (Poir.) Steud spiked with 0.1% w/w. Conclusion The developed assay is able to detect tyrosinase inhibitors present in complex matrixes in a reproducible way. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 25 March 2015 | 6:19 am

Rapid Identification of Steroidal Saponins in Trillium Tschonoskii Maxim by Ultraperformance Liquid Chromatography Coupled to Electrospray Ionisation Quadrupole Time-of-Flight Tandem Mass Spectrometry

Introduction Steroidal saponins in Trillium tschonoskii Maxim have many biological activities, including immunological regulation and anti-tumour. Comprehensive ingredient identification is critical for understanding its pharmacological mechanism and establishing quality control protocols. However, it is a challenging problem because of the complexity of steroidal saponins. Objectives To develop a UPLC–MS method for identifying and characterising steroidal saponins in the root and rhizome of T. tschonoskii. Methods Methanolic extracts of T. tschonoskii were analysed by using ultraperformance liquid chromatography coupled to electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (UPLC–ESI/QTOF/MS). The UPLC experiments were performed by means of a reversed-phase C18-column and a binary mobile phase system consisting of water and acetonitrile with formic acid under gradient elution conditions. For the UPLC–MS measurements, positive and negative ion modes were used in order to obtain better tandem mass spectra and high-resolution mass spectra. Results Based on retention times, accurate mass and mass spectrometric fragmentation, a total of 31 saponins distributed over eight steroidal aglycone skeletons were identified or tentatively elucidated from T. tschonoskii. Conclusion The UPLC–ESI/QTOF/MS method has proven to be a powerful tool for rapid identification of steroidal saponins in T. tschonoskii without tedious and time-consuming isolation of pure constituents. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 21 March 2015 | 3:04 pm

Comprehensive Two-dimensional Gas Chromatography Time-of-flight Mass Spectrometry to Assess the Presence of ?,?-Trehalose and Other Disaccharides in Apple and Peach

Introduction Carbohydrates are important constituents in fruits. Among the carbohydrates, disaccharides have rarely been studied in apple and peach. Indeed, the abiotic stress biomarker and preservation agent ?,?-trehalose is a disaccharide. Objectives To establish a comprehensive method based on two-dimensional gas chromatography combined with time-of-flight MS detection (GC?×?GC–ToF/MS) to analyse the disaccharide composition of apple and peach. Methods The sample preparation was based on aqueous-methanolic extraction of the analytes, followed by oxime formation and trimethylsilylation of the disaccharides. First, three columns were tested with standards on the one-dimensional system. Next, to perform the sample analysis using GC?×?GC–MS (which offers significant advantages over conventional GC because it allows higher separation efficiencies), various column configurations were assessed on the two-dimensional system to obtain enhanced separation and low detection limits. The column sets tested included non-polar/semi-polar, semi-polar/polar and polar/non-polar. Results Using the method that proved to be more efficient, namely the method developed with the semi-polar/non-polar configuration, ten disaccharides were identified, based on analytical standards, retention index and mass spectra. These compounds were quantified in several varieties of apple and peach fruit using the developed GC?×?GC method and linear curve calibration, resulting in substantial differences among the fruits. However, cultivars within the fruits exhibited no significant differences. Conclusion The proposed method allowed for the identification and quantification of several disaccharides in apple and peach, including the biomarker ?,?-trehalose. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 19 March 2015 | 8:56 am

Quantitative Determination of Secoiridoids and Phenylpropanoids in Different Extracts of Ligustrum Vulgare L. Leaves by a Validated HPTLC–Photodensitometry Method

Introduction The genus Ligustrum (Oleaceae) is distributed in Europe and Asia (south China and Korea), where it is used to prevent hypertension, sore throats, inflammation and diabetes. The main groups of compounds in extracts of Ligustrum vulgare are biologically active secoiridoids and phenylpropanoids. Objectives The aim of the study was primarily the development and validation of a HPTLC–photodensitometry method for separation and determination of secoiridoids (oleacein, oleuropein) and phenylpropanoids (echinacoside) in different extracts prepared from leaves of L. vulgare. A secondary issue was the quantitative screening of oleacein, oleuropein and echinacoside in extracts from leaves collected at different stages of plant growth (from May to September). Methods A HPTLC–photodensitometry method was developed and validated for quantification of oleuropein, oleacein and echinacoside in plant extracts (aqueous and ethanolic extract, decoction, infusion). Silica gel was used as the stationary phase and dichloromethane:methanol:formic acid:water (80:25:1.5:4, v/v/v/v) as the mobile phase. Results The HPTLC–photodensitometry method developed for quantification of oleacein, oleuropein and echinacoside was specific, accurate and precise. The presence of oleacein was detected in aqueous extracts, whereas oleuropein was present, in particular, in ethanolic extracts, decoctions and infusions. Echinacoside was detected in all the extracts prepared. The content of secoiridoids was variable from May to September, whereas the amount of echinacoside increased in this term. Conclusion The developed and validated HPTLC–photodensitometry method allowed performing fast screening of quantitative profiles of oleacein, oleuropein and echinacoside in preparations of privet leaves. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 19 February 2015 | 6:56 am

Discriminating Lamiophlomis rotata According to Geographical Origin by 1H-NMR Spectroscopy and Multivariate Analysis

Introduction Lamiophlomis rotata (Duyiwei) is a folk herbal medicine that traditionally has been used in China as a hemostatic agent. Raw plant materials used for medicinal products from different geographical regions are often inconsistent in chemical composition. Metabolic fingerprinting provides a new approach for distinguishing the geographical origins of L. rotata. Objective To identify metabolites that contribute to the different geographical regions of L. rotata samples. Methods Lamiophlomis rotata metabolomics were performed by 1H-nuclear magnetic resonance (NMR) spectroscopy and multivariate statistical analyses. The L. rotata metabolic profile was prepared for NMR measurements using methanol-d4 solvent. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to analyse the L. rotata 1H-NMR spectroscopy data. Results Nine iridoid glycosides, one flavonoid and three phenylpropanoid glycosides were detected in L. rotata by 1H-NMR spectroscopy. 1H-NMR measurements and multivariate analysis were used to successfully discriminate samples from three different locations. Conclusion The NMR-based analysis of L. rotata is a more comprehensive approach than traditional chromatographic methods. Simple sample preparation, rapidity and reproducibility of are additional advantages of NMR analysis. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 19 February 2015 | 6:55 am

Root Cause Analysis of Quality Defects Using HPLC–MS Fingerprint Knowledgebase for Batch-to-batch Quality Control of Herbal Drugs

Introduction The batch-to-batch quality consistency of herbal drugs has always been an important issue. Objectives To propose a methodology for batch-to-batch quality control based on HPLC–MS fingerprints and process knowledgebase. Methods The extraction process of Compound E-jiao Oral Liquid was taken as a case study. After establishing the HPLC–MS fingerprint analysis method, the fingerprints of the extract solutions produced under normal and abnormal operation conditions were obtained. Multivariate statistical models were built for fault detection and a discriminant analysis model was built using the probabilistic discriminant partial-least-squares method for fault diagnosis. Results Based on multivariate statistical analysis, process knowledge was acquired and the cause–effect relationship between process deviations and quality defects was revealed. The quality defects were detected successfully by multivariate statistical control charts and the type of process deviations were diagnosed correctly by discriminant analysis. Conclusion This work has demonstrated the benefits of combining HPLC–MS fingerprints, process knowledge and multivariate analysis for the quality control of herbal drugs. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 19 February 2015 | 6:55 am

Densitometric Validation and Optimisation of Polyphenols in Ocimum sanctum Linn by High Performance Thin-layer Chromatography

Introduction Ocimum sanctum Linn (Sanskrit: Tulasi; family: Libiaceae), popularly known as holy basil or Ocimum teinufolium, is found throughout the semitropical and tropical parts of India. In Ayurveda, Tulasi has been well known for its therapeutic potentials. Objective To optimise and develop a standard method to quantify seven polyphenols simultaneously by HPTLC. Methods A three-level factor Box–Behnken statistical design was used for optimisation, where extraction time (min), temperature (°C) and methanol:water ratio (% v/v) are the independent variables with polyphenols as the dependent variable. The separation was archived on a silica-gel 60?F254 HPTLC plate using toluene:ethyl acetate:formic acid:methanol (3:3:0.8:0.2?v/v) as the mobile phase. Densitometric analysis of polyphenols was carried out in the absorbance mode at 366?nm. Results The quantification of polyphenols was carried out based on peak area with a linear calibration curve at concentration ranges of 60–240, 20–200, 100–1600, 40–200, 200–1400, 10–160, 200–1400, 100–5000?ng/band for caffeic acid, ellagic acid, rutin, kaempferol, catechin, quercetin, eupalitin and epicatechin respectively. The method was validated for peak purity, precision, accuracy, limit of detection (LOD) and quantification (LOQ). Method specificity was confirmed using the retention factor value and visible spectra correlation of marker compounds. Conclusions A validated HPTLC method was newly developed for simultaneous quantification of seven polyphenols in an Ayurvedic preparation of O. sanctum. The proposed method is simple, precise, specific, accurate, cost-effective, less time consuming and has the ability to separate the polyphenols from other constituents. Copyright © 2015 John Wiley & Sons, Ltd.

Posted on 10 February 2015 | 7:17 am

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