Phytochemical profile of fresh and senescent leaves due to storage for Ficus deltoidea

This study was carried out to investigate the phytochemical profiles of plant leaves, namely fresh and senescent leaves from Ficus deltoidea upon storage at room temperature. The phytochemical profile is of great importance, since this herb is widely used as traditional herbal medicine. Both chromatographic and mass spectrometric profiles of the plant extracts were fingerprinted using a high sensitivity hyphenated system consisting of liquid chromatography integrated with tandem mass spectrometer. Identical extraction protocol was used to extract phytochemicals from both leaf samples using 50% v/v methanolic aqueous solvent system. The plant extracts were determined for their total phenolic and flavonoid contents, as well as their antioxidant capacities based on radical scavenging, ferrous chelation, and ferric reducing power assays. The results showed that senescent leaf extracts exhibited higher antioxidant capacity than fresh leaf samples. This observation could be due to the higher number of phenolic compounds in the senescent leaf samples. The senescence of postharvest leaves was likely to consume organic acids including phenolic acids in the defense mechanism against the drought stress. However, flavonoids, particularly flavones and isoflavones, were abundant in the senescent leaf extracts. The findings may explain the significant pharmacological properties reported by previous investigators.     

High-performance liquid chromatography with atmospheric pressure chemical ionization and electrospray ionization mass spectrometry for analysis of Angelica sinensis

An HPLC-PAD-API/MS method for analysing the chemical constituents of Angelica sinensis (A. sinensis) has been developed. ESI and APCI spectra, in both positive ion (PI) and negative ion (NI) modes, provided very useful information concerning the molecular weights of detected compounds. By comparing the retention times, UV spectra, mass spectra and molecular weights of detected compounds with those published in literature, 15 constituents of A. sinensis could be tentatively identified. This technique involving combined MS information may provide an objective, reliable and rapid analytical method for the quality control and database research of traditional Chinese medicines.     

Solid-phase extraction and HPLC-MS profiling of pyrrolizidine alkaloids and their N-oxides: a case study of Echium plantagineum

Pyrrolizidine alkaloids and their N-oxides can be extracted from the dried methanolic extracts of plant material using dilute aqueous acid. The subsequent integration of solid-phase extraction (with a strong cation exchanger) of the alkaloids and N-oxides from the aqueous acid solution, together with analysis using HPLC-ESI/MS, provides a method for the simultaneous profiling of the pyrrolizidine alkaloids and their N-oxides in plant samples and the collection of useful structural data as an aid in their identification. The N-oxide character of the analytes may be confirmed by treating analytical samples with a redox resin and observing the formation of the corresponding parent pyrrolizidine alkaloids. The present case study of Echium plantagineum highlighted a higher ratio of N-oxides to the parent tertiary bases than has been previously reported. Furthermore, a higher proportion of acetylated pyrrolizidine-N-oxides was observed in the flower heads relative to the leaves. Six pyrrolizidine alkaloids or pyrrolizidine-N-oxides, not previously reported from E. plantagineum, were tentatively identified on the basis of MS and biogenetic considerations. Three of these, 3'-O-acetylintermedine/lycopsamine, leptanthine-N-oxide and 9-O-angelylretronecine-N-oxide, have been reported elsewhere, whilst three others, 3'-O-acetylechiumine-N-oxide, echimiplatine-N-oxide and echiuplatine-N-oxide, appear unreported from any other source.     

Determination of carbohydrates in medicinal plants--comparison between TLC, mf-MELDI-MS and GC-MS

Introduction – Quality control in the pharmaceutical and phytopharmaceutical industries requires fast and reliable methods for the analysis of raw materials and final products. Objective – This study evaluates different analytical approaches in order to recognise the most suitable technique for the analysis of carbohydrates in herbal drug preparations. Methodology – The specific focus of the study is on thin‐layer chromatography (TLC), gas chromatography (GC), and a newly developed mass spectrometric method, i.e. matrix free material enhanced laser desorption/ionisation time of flight mass spectrometry (mf‐MELDI‐MS). Samples employed in the study were standards and microwave‐assisted water extracts from Quercus. Results – TLC analysis proved the presence of mono‐, di‐ and trisaccharides within the biological sample and hinted at the existence of an unknown carbohydrate of higher oligomerisation degree. After evaluation of different derivatisation techniques, GC‐MS confirmed data obtained via TLC for mono‐ to trisaccharides, delivering additionally quantified values under a considerable amount of time. A carbohydrate of higher oligomerisation degree could not be found. The application of mf‐MELDI‐MS further confirmed the presence of carbohydrates up to trisaccharides, also hinting at the presence of a form of tetrasaccharide. Besides this information, mf‐MELDI‐MS delivered further data about other substances present in the extract. Quantitative determination resulted in 1.750, 1.736 and 0.336 mg/mL for glucose, sucrose and raffinose respectively. Conclusion – Evaluation of all three techniques employed, clearly proved the heightened performance of mf‐MELDI‐MS for the qualitative analysis of complex mixtures, as targets do not need modification and analysis requires only a few minutes. In addition, GC‐MS is suitable for quantitative analysis. Copyright © 2011 John Wiley & Sons, Ltd.     

Validated Method for Strigolactone Quantification by Ultra High‐Performance Liquid Chromatography – Electrospray Ionisation Tandem Mass Spectrometry Using Novel Deuterium Labelled Standards

Introduction

Strigolactones (SLs) are important plant hormones. They are difficult to analyse because they occur in very small concentrations especially in comparison with other plant hormones and other substances can interfere with their detection.

Objective

To develop a procedure for the extraction, purification and quantification of SLs from plant roots.

Methodology

Samples were prepared by extraction of plant root tissues with ethyl acetate. Then the extracts were further purified with silica column chromatography. The natural SLs in the final extracts were quantified using novel deuterium labelled SLs. The results of the methodology were compared with those of the procedure of Yoneyama and coworkers.

Results

This procedure required about 1‐g root samples to detect and quantify simultaneously the SLs (orobanchyl acetate and fabacyl acetate) concentration with high reliability.

Conclusion

A method was developed for determining endogenous fabacyl acetate and orobanchyl acetate in plant tissue based on novel deuterium labelled standards. A method of orobanchol quantification using a synthetic SL GR24 as internal standard was proposed. Copyright © 2017 John Wiley & Sons, Ltd.     

Product and contaminant measurement in bioprocess development by SELDI‐MS

Bioprocesses for therapeutic protein production typically require significant resources to be invested in their development. Underlying these efforts are analytical methods, which must be fit for the purpose of monitoring product and contaminants in the process. It is highly desirable, especially in early‐phase development when material and established analytical methods are limiting, to be able to determine what happens to the product and impurities at each process step with small sample volumes in a rapid and readily performed manner. This study evaluates the utility of surface‐enhanced laser desorption ionization mass spectroscopy (SELDI‐MS), known for its rapid analysis and minimal sample volumes, as an analytical process development tool. In‐process samples from an E. coli process for apolipoprotein A‐IM (ApoA‐IM) manufacture were used along with traditional analytical methods such as HPLC to check the SELDI‐MS results. ApoA‐IM is a naturally occurring variant of ApoA‐I that appears to confer protection against cardiovascular disease to those that carry the mutated gene. The results show that, unlike many other analytical methods, SELDI‐MS can handle early process samples that contain complex mixtures of biological molecules with limited sample pretreatment and thereby provide meaningful process‐relevant information. At present, this technique seems most suited to early‐phase development particularly when methods for traditional analytical approaches are still being established. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A RP‐HPLC‐DAD‐APCI/MSD method for the characterisation of medicinal Ericaceae used by the Eeyou Istchee Cree First Nations

Introduction – Ericaceae medicinal plants are traditionally used by the Eeyou Istchee Cree and other northern peoples of North America to treat type 2 diabetic symptoms. Because of the importance of phenolics as potential cures for degenerative diseases including type 2 diabetes, an analytical method was developed to detect them in the leaf extracts of 14 Ericaceae plants. Objective – To develop an optimised method which is applicable to a relatively large number of Ericaceae plants using their leaf extracts. For this purpose phenolics with a wide range of polarity, including a glucosylated benzoquinone, two phenolic acids, three flavanols, a flavanone, a flavone and five flavonols, were included in this study. Methodology – Characterisation of phytochemicals in extracts was undertaken by automated matching to the UV spectra to those of an in house library of plant secondary metabolites and the authentication of their identity was achieved by reversed phase‐high‐performance chromatography–diode array detection–atmospheric pressure chemical ionisation/mass selective detection. Results – Twenty‐six phenolics were characterised within 26 min of chromatographic separation in 80% ethanol extracts of 14 Ericaceae plants. The calibration curves were linear within 0.5–880 µg/g dry mass of the plant with regression values better than 0.995. The limits of detection ranged from 0.3 for µg/mL for (+)‐catechin to 2.6 µg/mL for chlorogenic acid. This is a first study dealing with relatively large number of Ericaceae extracts and is applicable to other plants of same family. Copyright © 2010 John Wiley & Sons, Ltd.     

Isolation and characterisation of selected germander diterpenoids from authenticated Teucrium chamaedrys and T. canadense by HPLC, HPLC-mS and NMR

Teucrium species, such as germander, are rich in neo-clerodane diterpenoids and have been used in traditional folk medicine for their stimulant, diuretic, antipyretic and antiseptic properties. However, the furano neo-clerodane diterpenoids present in germander have been implicated in the in vivo hepatotoxicity of this botanical. In this study, authenticated germander (Teucrium chamaedrys L. and Teucrium canadense L.) was used as the source material. Methanol extracts of powdered plant mate rial were prepared and analysed by HPLC using Synergi Max-RP columns with monitoring at 220 nm. Limited amounts of teucrin A and other diterpenoid standards were analysed on a Synergi Max-RP column in order to determine their retention times and to generate calibration curves. The same standards were subjected to concurrent mass spectral analysis. Teucrin A and diterpenoids such as dihydroteugin, teuflin, teuflidin and teucvidin were tentatively identified in the plant extracts by HPLC-MS and 1H-NMR experiments. For the isolation of teucrium diterpenoids on a semipreparative scale, a solid-phase extraction method was developed for the first time using styrene divinylbenzene and strata-X sorbents for teucrin A and teuflin, respectively. Semi-preparative HPLC of the methanol extract of the powdered aerial parts of Teucrium plants was carried out on a semipreparative Synergi Max-RP column with photodiode array detection in order to confirm the identities of some diterpenoids by HPLC-MS and NMR.     

Development of an HPLC-PAD-MS assay for the identification and quantification of major phenolic edelweiss (Leontopodium alpium Cass.) constituents

The analytical assessment of edelweiss (Leontopodium alpinum) herb extracts, used in traditional alpine medicine, has resulted in the development of a HPLC-PAD-MS method that allows baseline separation of almost all constituents. Peak assignment of 14 analytes was achieved by comparison of retention times, UV and mass spectra with those of reference compounds either commercially available (luteolin, apigenin and chlorogenic acid) or isolated from edelweiss plants by column chromatography. Ten of the isolated analytes were identified as the known natural products: quercetin-3-O-beta-D-glucoside, luteolin-7-O-beta-D-glucoside, luteolin-3'-O-beta-D-glucoside, luteolin-4'-O-beta-D-glucoside, apigenin-7-O-beta-D-glucoside, 6-hydroxy-luteolin-7-O-beta-D-glucoside, luteolin-7,4'-di-O-beta-D-glucoside, chrysoeriol-7-O-beta-D-glucoside, leontopodic acid and 3,5-dicaffeolyquinic acid. One analyte, 3,4,5-tri-(E)-caffeoly-D-glucaric acid proved to be a new natural product and was named leontopodic acid B. Structure elucidation was carried out by means of MS and NMR spectroscopy in all cases. The aerial plant parts of L. alpinum (capitula, inflorescence leaves, stems, stem leaves and leaves of the basal rosette) showed variable amounts of the above-mentioned constituents, although qualitative differences were not observable.     

An acylated flavonol glycoside and hydrolysable tannins from Callistemon lanceolatus flowers and leaves

Introduction –  Callistemon lanceolatus DC. (Myrtaceae) is a plant rich in polyphenols, and is used as anticough, antibronchitis and insecticide in folk medicine. Because of the biological importance of plant polyphenols, particularly tannins, a phytochemical study was of interest to investigate the constitutive poyphenols in the extracts of flowers and leaves. Objective –  To avoid time‐consuming methodology for isolation of a complex mixture of known metabolites, HPLC‐ESI/MS was employed for fast picking up of the new compounds followed by identification of the structures with UV and one‐ and two‐dimensional NMR. Methodology –  Flowers and leaves were separately extracted with hot aqueous methanol under reflux (70°C). Pre‐isolation of the total extracts was achieved through column chromatographic fractionation on polyamide with water–methanol for gradient elution. The main fractions were purified using repeated column chromatography on cellulose and/or Sephadex LH‐20 with suitable eluents. HPLC‐ESI/MS analyses were carried out in the single ion monitoring (SIM) and negative ion modes. The pure compounds in methanol–water (1:1) were analysed by direct infusion ESI/MS. Final structure elucidation was obtained by one‐ and two‐dimensional NMR. Results –  Two new metabolites namely quercetin 3‐O‐β‐D‐glucuronopyranoside n‐butyl ester ( 1 ) and n‐butylgallate 4‐O‐(2′,6′‐di‐O‐galloyl)‐β‐d ‐glucopyranoside ( 4 ) along with nine known ones were identified from the aqueous methanol extracts of flowers and leaves. Conclusion –  The study has shown that Callistemon lanceolatus is rich in polyphenols. HPLC‐ESI/MS may be used, in negative ion mode, as an efficient and rapid analytical tool for investigating complex plant extracts. Copyright © 2008 John Wiley & Sons, Ltd.     

A Comprehensive Characterisation of Rosemary tea Obtained from Rosmarinus officinalis L. Collected in a sub‐Humid Area of Tunisia

Introduction

Rosemary (Rosmarinus officinalis L.) is an aromatic plant common in Tunisia and it is widely consumed as a tea in traditional cuisine and in folk medicine to treat various illnesses. Currently, most research efforts have been focused on rosemary essential oil, alcoholic and aqueous extracts, however, little is reported on rosemary infusion composition.

Objective

To investigate compounds present in rosemary tea obtained from Rosmarinus officinalis L. collected in a sub‐humid area of Tunisia in order to assess whether the traditional rosemary tea preparation method could be considered as a reference method for rosemary's compounds extraction.

Methodology

Qualitative characterisation of Rosmarinus officinalis tea obtained after rosemary infusion in boiled water was determined by high performance liquid chromatography coupled with electrospray ionisation quadrupole time‐of‐flight mass spectrometry (HPLC‐ESI‐QTOF‐MS). Quantitative analysis relies on high performance liquid chromatography with diode array detector (HPLC‐DAD).

Results

Forty‐nine compounds belonging to six families, namely flavonoids, phenolic acids, phenolic terpenes, jasmonate, phenolic glycosides, and lignans were identified. To the best of the authors' knowledge eucommin A is characterised for the first time in rosemary. Rosmarinic acid (158.13 μg/g dried rosemary) was the main compound followed then by feruloylnepitrin (100.87 μg/g) and luteolin‐3′‐O‐(2″‐O‐acetyl)‐β‐d ‐glucuronide (44.04 μg/g). Among quantified compounds, luteolin‐7‐O‐rutinoside was the compound with the lowest concentration.

Conclusion

The infusion method allows several polyphenols present in rosemary tea to be extracted, therefore it could be a reference method for rosemary's compounds extraction. Moreover, traditional Tunisian Rosmarinus officinalis tea consumption is of interest for its rich phenolic content. Copyright © 2017 John Wiley & Sons, Ltd.     

Analytical characterisation of crude extracts from an African Ancistrocladus species using high-performance liquid chromatography and capillary electrophoresis coupled to ion trap mass spectrometry

The analysis by HPLC, CE and CE-MS/MS of root bark extracts of a, so far undescribed, Central-African Ancistrocladus species (family Ancistrocladaceae) is described. Owing to the complexity of the extract, the application of reversed-phase HPLC resulted in a partially incomplete separation of the naphthylisoquinoline alkaloids, whilst CE using a non-aqueous buffer proved to be a very valuable complementary method for a first characterisation of the crude extract. By performing additional CE-MS/MS experiments, in combination with parallel isolation studies and structural elucidation using conventional methods, six alkaloidal substances present in the plant could be identified.     

A single HPLC-PAD-APCI/MS method for the quantitative comparison of phenolic compounds found in leaf, stem, root and fruit extracts of Vaccinium angustifolium

A method was developed for the analysis of Vaccinium angustifolium Ait. (Lowbush blueberry), which is a widely used natural health product, particularly for the treatment of diabetic symptoms. While the anthocyanin content of the fruit has been well characterized, the chemistry of the vegetative parts used in supportive therapy for diabetes has been largely ignored. Using a metabolomics-based approach for compound identification with an emphasis on phenolic metabolites, a single HPLC-PAD-APCI/ MS method was developed for the separation and quantitation of the major metabolites found in the 95% ethanol extracts of leaf, stem, root and fruit. The leaf extract contained high concentrations of chlorogenic acid (approximately 100 microg/mg extract) and a variety of quercetin glycosides that were also detected in the fruit and stem extracts. Flavan-3-ol monomers (+)-catechin and (-)-epicatechin were found in all plant parts but their procyanidin dimers were exclusively identified in the stem and root. The accuracy and precision of the presented method were corroborated by low intra- and inter-day variations in quantitative results in all plant part extracts. Further validation of the extraction and analytical protocols focused on identified compounds with reputed anti-diabetic activity, revealing recoveries greater than 80% and detection limits of 0.12-2.73 microg/mL.     

Plumbagin recovery from field specimens of Drosophyllum lusitanicum (L.) link

The naphthoquinone plumbagin has a broad spectrum of biological activities. The aim of this study was to investigate the efficiency of two extraction methods (Soxhlet and ultrasound-assisted extraction) and three solvents (methanol, chloroform and hexane) to recover plumbagin from fresh and dried tissues of field specimens of Drosophyllum lusitanicum (L.) Link. The highest extraction yields were obtained with methanol as solvent and using fresh plant material. The obtained extracts were analysed by gas chromatography with mass spectrometric detection and plumbagin was the major compound present. Plumbagin was quantified in the extracts using the external standard methodology. The results obtained showed that the best recoveries of plumbagin were attained using fresh plant material and there were no significant differences between Soxhlet and ultrasound-assisted extraction. Moreover, hexane proved to be the more appropriate solvent for the extraction of plumbagin, providing high recoveries and the most concentrated extracts, yielding 2.42 mg of plumbagin per gram of plant material with the highest degree of purity. This method is a simple and efficient one to extract large amounts of plumbagin from D. lusitanicum field specimens.     

Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data

The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data.Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared.This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.     

Optimized proteomic analysis of a mouse model of cerebellar dysfunction using amine-specific isobaric tags

Recent proteomic applications have demonstrated their potential for revealing the molecular mechanisms underlying neurodegeneration. The present study quantifies cerebellar protein changes in mice that are deficient in plasma membrane calcium ATPase 2 (PMCA2), an essential neuronal pump that extrudes calcium from cells and is abundantly expressed in Purkinje neurons. PMCA2-null mice display motor dyscoordination and unsteady gait deficits observed in neurological diseases such as multiple sclerosis and ataxia. We optimized an amine-specific isobaric tags (iTRAQ)-based shotgun proteomics workflow for this study. This workflow took consideration of analytical variance as a function of ion signal intensity and employed biological repeats to aid noise reduction. Even with stringent protein identification criteria, we could reliably quantify nearly 1000 proteins, including many neuronal proteins that are important for synaptic function. We identified 21 proteins that were differentially expressed in PMCA2-null mice. These proteins are involved in calcium homeostasis, cell structure and chromosome organization. Our findings shed light on the molecular changes that underlie the neurological deficits observed in PMCA2-null mice. The optimized workflow presented here will be valuable for others who plan to implement the iTRAQ method.     

Phytochemical evaluation and anticancer activity of rambutan (Nephelium lappaceum) fruit endocarp extracts against human hepatocellular carcinoma (HepG-2) cells

The current investigation was taken to screen the phytoconstituents present in fruit endocarp various extracts of Nephelium lappaceum commonly called as Rambutan fruit and its anticancer property against human hepatocellular carcinoma (HepG-2) cells. Different analytical techniques including qualitative phytochemical analysis, cell viability assay (MTT), apoptotic nuclear staining (DAPI), DNA fragmentation assay, Attenuated total reflection (ATR) and Gas chromatography–mass spectrometry (GC–MS) spectral analysis were carried out. ATR and GC–MS study revealed the presence of functional groups and 9 compounds, respectively in methanol endocarp extract. The results obtained depicts that methanol endocarp extract profoundly controlled cell proliferation and caused shrinkage of HepG-2 cells from polygonal to spherical shape. DAPI staining revealed that methanol endocarp extract caused increased fragmentation of nucleus and DNA fragmentation, which can be taken as a sign of apoptosis. The anticancer potential of methanol fruit endocarp extract of Nephelium lappaceum than other extracts and could be used successfully in future drug delivery systems and other biomedical concerns.     

抗抑郁药用植物研究进展

近年来,虽然药用植物被用于临床治疗精神紊乱和行为异常等相关性疾病,如:抑郁、焦虑、癫痫、记忆力衰退、失眠、老年痴呆和药物中毒等,但是,有关药用植物抗抑郁作用的研究性报道较少,且不够系统。本综述主要归纳和总结了具有抗抑郁作用的药用植物及其活性提取物,包括药用植物粗提物和单体活性成分等天然产物,为充分开发利用我国药用植物资源以及民间传统用药提供科学依据,促进高效安全抑郁症治疗方法的研究。     

Rapid selective screening and determination of ephedrine alkaloids using GC-MS footnote mark

Ephedra (ma huang) has been widely used as an herb or herbal extract in both traditional Chinese medicine and Western world dietary supplements. The effects of Ephedra have been attributed to a series of six ephedrine alkaloids including ephedrine and pseudoephedrine. A GC-MS method for the ephedrine alkaloids is described which couples ammoniacal chloroform as the extraction solvent with a two-stage derivatisation scheme. This scheme produces the O-trimethylsilyl, N-trifluoracetyl derivatives (O-TMS, N-TFA) for the primary and secondary amine alkaloids, and the O-TMS derivatives for the tertiary amine alkaloids. Relatively clean extracts are obtained from complex matrices, and the six ephedrine alkaloids are effectively separated and identified. This approach was also evaluated for quantitative analysis, and was shown to provide quantitative results for ephedrine and pseudoephedrine, and good estimates for the four minor alkaloids. Figures of merit are presented for linearity, detection limits, precision and accuracy. We have applied this approach to the rapid screening and profiling of the ephedrine alkaloids in whole Ephedra plants, liquid plant extracts, dried powder plant extracts and a variety of Ephedra-containing dietary supplements.     

DNA宏条形码技术在食草动物食性研究中的应用

随着测序技术的快速发展,整合DNA条形码和高通量测序的DNA宏条形码技术已经成为当前研究热点之一,在食草动物的食性鉴定中有很大潜力。放牧动物食性研究是动物营养学和草地生态学领域的重要研究内容。而与传统食性研究方法相比,宏条形码技术可通过对植物DNA条形码的高通量测序,获得样本中的物种组成进而分析动物食性。介绍了传统食性分析手段的局限,重点综述了DNA宏条形码技术的产生、操作原理以及在食草类动物食性鉴定领域中的应用,同时还简述了可能存在的挑战,并对该技术今后的发展方向进行了展望。