Phytochemistry: Current Research Articles
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On this page considered biochemistry journals:
Phytochemistry Reviews - published by
Springer -
Phytochemical Analysis - published by
Wiley-Interscience -
Current research articles of the mentioned
journals:
Use of digitoxin and digoxin as internal standards in HPLC analysis of triterpene saponin-containing extracts
Saponins are widely distributed complex plant glycosides possessing a variety of structure-dependent bioactivities. Quantitation of individual saponins is difficult due to lack of available standards, mainly as a consequence of purification difficulties. Determination of total saponin content can be problematic, often relying on non-specific methods based on butanol solubility, haemolytic activity or formation of coloured derivatives.To develop a general quantitative method based on the use of the readily available cardenolides, digitoxin (1) and digoxin (2), as internal standards in an HPLC-PAD-based analysis.The cardenolides were run at a variety of concentrations to establish linearity and reproducibility of detector response and then evaluated as internal standards for quantitation of triterpene saponins in several plant-derived extracts by HPLC-PAD. Mixtures of saponins, largely freed from other extractables, were obtained by fractionation of total extracts on solid phase extraction columns (SPE) employing a water-methanol gradient and used for construction of calibration curves. Saponin identification and structural information was obtained via a single quadrupole mass detector using electrospray ionisation in negative ion mode (ESI-).Saponin contents in six samples from five species were determined and compared with literature results and a gravimetric method based on butanol-water partitioning. Results were generally consistent with literature reports and superior to gravimetric butanol-water partitioning.Digitoxin and digoxin are useful as internal standards in HPLC estimation of saponin content. Saponins from different species having similar structures and molecular weights afford similar calibration curves. Copyright © 2008 John Wiley & Sons, Ltd.
Immunochromatographic assay for the detection of pseudojujubogenin glycosides
Bacopa monnieri contains pseudojujubogenin glycosides as pharmacologically active compounds. In order to screen large numbers of plant samples for the presence of pseudojujubogenin glycosides, a rapid and simple assay system is required for application to small quantities of test materials. Immunoassays using monoclonal antibodies could be useful for the determination of small quantities of pseudojujubogenin glycosides in plant extracts.The objective of this work was to develop a simple method for the detection of pseudojujubogenin glycosides by the immunochromatographic strip test using anti-bacopaside I monoclonal antibody.The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of a colloidal gold particle coated with the respective anti-bacopaside I MAb. The capture reagent was a bacopaside I-human serum albumin conjugate immobilised onto a test strip membrane.The sample containing pseudojujubogenin glycosides and the detection reagent were incubated with the immobilised capture reagent. The glycosides in the sample competed in binding to the limited amount of antibodies in the detection reagent with the immobilised bacopaside I-HSA conjugates and, hence, positive samples showed no colour in the capture spot zone. The detection limit for the strip test was 125 ng/mL.The assay system was found to be useful as a rapid and simple screening method for the detection of pseudojujubogenin glycosides in plants. Copyright © 2008 John Wiley & Sons, Ltd.
Simultaneous determination of 76As, 122Sb and 153Sm in Chinese medicinal herbs by epithermal neutron activation analysis
Optimal conditions for the simultaneous determination of As, Sb and Sm in Chinese medicinal herbs using epithermal neutron activation analysis were investigated. The minimum detectable concentrations of 76As, 122Sb and 153Sm in lichen and medicinal herbs depended on the weight of the irradiated sample, and irradiation and decay durations. Optimal conditions were obtained by wrapping the irradiated target with 3.2 mm borated polyethylene neutron filters, which were adopted to screen the original reactor fission neutrons and to reduce the background activities of 38Cl, 24Na and 42K. Twelve medicinal herbs, commonly consumed by Taiwanese children as a diuretic treatment, were analysed since trace elements, such as As and Sb, in these herbs may be toxic when consumed in sufficiently large quantities over a long period. Various amounts of medicinal herbs, standardised powder, lichen and tomato leaves were weighed, packed into polyethylene bags, irradiated and counted under different conditions. The results indicated that about 350 mg of lichen irradiated for 24 h and counted for 20 min following a 30-60 h decay period was optimal for irradiation in a 1011 n/cm s epithermal neutron flux. The implications of the content of the studied elements in Chinese medicinal herbs are discussed. Copyright © 2008 John Wiley & Sons, Ltd.
A validated reverse-phase HPLC analytical method for the quantification of phenolic compounds in Baccharis dracunculifolia
Baccharis dracunculifolia, which has great potential for the development of new phytotherapeutic medicines, is the most important botanical source of the southeastern Brazilian propolis, known as green propolis on account of its color.To develop a reliable reverse-phase HPLC chromatographic method for the analysis of phenolic compounds in both B. dracunculifolia raw material and its hydroalcoholic extracts.The method utilised a C18 CLC-ODS (M) (4.6 × 250 mm) column with nonlinear gradient elution and UV detection at 280 nm. A procedure for the extraction of phenolic compounds using aqueous ethanol 90%, with the addition of veratraldehyde as the internal standard, was developed allowing the quantification of 10 compounds: caffeic acid, coumaric acid, ferulic acid, cinnamic acid, aromadendrin-4[prime]-methyl ether, isosakuranetin, drupanin, artepillin C, baccharin and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran acid.The developed method gave a good detection response with linearity in the range 20.83-800 µg/mL and recovery in the range 81.25-93.20%, allowing the quantification of the analysed standards.The method presented good results for the following parameters: selectivity, linearity, accuracy, precision, robustness, as well as limit of detection and limit of quantitation. Therefore, this method could be considered as an analytical tool for the quality control of B. dracunculifolia raw material and its products in both cosmetic and pharmaceutical companies. Copyright © 2008 John Wiley & Sons, Ltd.
Application of Scion image software to the simultaneous determination of curcuminoids in turmeric (Curcuma longa)
Curcumin, desmethoxycurcumin and bisdesmethoxycurcumin are bioactive constituents of turmeric (Curcuma longa). Owing to their different potency, quality control of turmeric based on the content of each curcuminoid is more reliable than that based on total curcuminoids. However, to perform such an assay, high-cost instrument is needed.To develop a simple and low-cost method for the simultaneous quantification of three curcuminoids in turmeric using TLC and the public-domain software Scion Image.The image of a TLC chromatogram of turmeric extract was recorded using a digital scanner. The density of the TLC spot of each curcuminoid was analysed by the Scion Image software. The density value was transformed to concentration by comparison with the calibration curve of standard curcuminoids developed on the same TLC plate.The polynomial regression data for all curcuminoids showed good linear relationship with R2 > 0.99 in the concentration range of 0.375-6 µg/spot. The limits of detection and quantitation were 43-73 and 143-242 ng/spot, respectively. The method gave adequate precision, accuracy and recovery. The contents of each curcuminoid determined using this method were not significantly different from those determined using the TLC densitometric method.TLC image analysis using Scion Image is shown to be a reliable method for the simultaneous analysis of the content of each curcuminoid in turmeric. Copyright © 2008 John Wiley & Sons, Ltd.
Assessment of different sample processing procedures applied to the determination of melatonin in plants
Introduction - Melatonin, an indoleamine well known in vertebrates and structurally related to other important substances such as tryptophan or indole-3-acetic acid, is also present in the plant kingdom although its specific function(s) remain to be established. The emerging field of melatonin studies in plants has progressed very slowly, mainly due to the problems associated with melatonin quantification in plants.Objective - Two commonly used procedures for plant samples are compared. The analytical characteristics of both procedures are quantitatively presented using different solvents and small amounts of fresh biological material, and the respective recovery rates and quantitative limits are presented. Some improvements are suggested.Methodology - Two different sample extraction procedures were compared: a direct-sample extraction (DSE) and a homogenised- sample extraction (HSE). Melatonin was then determined in the respective plant samples by HPLC with fluorescence detection.Results - Using the DSE procedure, more than 94% melatonin was recovered from standard solutions, whereas levels higher than 93% were recovered from the spiked plant samples, with little difference between ethyl acetate and chloroform extractions. In the case of HSE, the recoveries of melatonin were approximately half and never higher than 55%. The ultrasonic treatment proposed in the DSE procedure showed different levels of efficiency (2-20%), depending on the sample.Conclusion - This study has established that, with the direct sample extraction procedure, higher recovery rates are obtained both in standard solutions and in plant samples. The straightforwardness and reproducibility of the extraction procedure is accompanied by the high sensitivity obtained with fluorescence detection. Copyright © 2008 John Wiley & Sons, Ltd.
New evidence for the molecular-chemical diversity of potato plant rhizodeposits obtained by pyrolysis-field Ionisation mass spectrometry
Introduction - Detailed descriptions of the molecular-chemical diversity in plant rhizodeposits are scarce. The vast majority of our knowledge is derived from a priori methods of analysis, such as GC-MS and HPLC.Objective - To analyse the composition of rhizodeposits from the potato cultivar Solanum tuberosum L. cv. Albatros by pyrolysis -field ionisation mass spectrometry (Py-FIMS) and to explain differences in relation to plant growth stage and photoperiod.Methodology - Potato (Solanum tuberosum L.) plants were grown in non-sterile, native soil under controlled environmental conditions (plant chamber). Rhizodeposit samples were collected by leaching during two different growth stages and after the physiological day- and night-cycle. All leachate samples were investigated by Py-FIMS. Mass spectrometric data were evaluated by multivariate statistics.Results - Screening of the rhizodeposits by Py-FIMS revealed a broad range of m/z signals. Low-molecular-weight substances of m/z 15-56 (8.1-18.6%), alkylaromatics (12.0-15.9%), phenols and lignin monomers (8.8-13.1%) and carbohydrates (6.0-11.2%) comprised the largest proportions of total ion intensity (TII). Mass signals with significantly different abundance at the various sampling dates were assigned to compound classes of carbohydrates, phenols and lignin monomers, lignin dimers, lipids, N-containing compounds, sterols, peptides and free fatty acids; these were supplemented by marker signals for N-acetylmuramic acid from bacterial cell walls and signal molecules for the regulation of secondary pathways such as 4-hydroxycinnamic acid and linolenic acid.Conclusion - Py-FIMS was well suited to detect the molecular-chemical diversity of potato plant rhizodeposits and, compared with traditional a priori analytical methods, provided detailed evidence for significant differences in the composition of rhizodeposits depending on growth stage and diurnal period. Copyright © 2008 John Wiley & Sons, Ltd.
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-[beta]-D-glucuronopyranoside n-butyl ester (1) and n-butylgallate 4-O-(2[prime],6[prime]-di-O-galloyl)-[beta]-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.
Chemical fingerprinting of Lawsonia inermis L. using HPLC, HPTLC and densitometry
Introduction - Lawsonia inermis L. is a natural red colouring agent, commonly named "Henna", which is used to dye skin and hair. The aim of this study was to evaluate the quality of L. inermis that is commercially available as a raw plant material or preparation in order to guarantee good quality products.Objective - To develop a simple protocol for the qualification of different samples labelled as L. inermis by using the HPTLC densitometry method and to identify possible adulterations with other plants.Methodology - Samples of leaves of L. inermis were extracted with methanol. Two chromatographic methods were developed to determine the chemical fingerprinting of L. inermis. The first was based on HPTLC identification followed by densitometric measurements at 337 nm. The second was based on RP-HPLC separation with gradient elution and photodiode array detection at 337 nm. Samples of Cassia obovata Collad., and Indigofera tinctoria L., were treated in the same way.Results - The simplicity of the sample preparation, and the possibility of analysing several samples of herbal products simultaneously in a short time, make HPTLC the method of choice. The HPTLC method was feasible for the comprehensive quality evaluation of herbal products. From the comparison of their "fingerprint", it was possible to detect substitution of plants that are different from those declared on the label.Conclusion - The HPTLC may be used as a rapid method by which to control the quality of raw plant materials and formulations based on the title plant. Copyright © 2008 John Wiley & Sons, Ltd.
Influence of the extraction mode on the yield of hyperoside, vitexin and vitexin-2[prime][prime]-O-rhamnoside from Crataegus monogyna Jacq. (hawthorn)
Introduction - The extract of Crataegus monogyna shows sedative, hypotensive, vasodilator and cardio-tonic actions. Although several papers dealing with the extraction of metabolites from Crataegus have been published, the plant productivity in terms of bioactive compounds is not easily understandable as yet.Objective - To investigate the influence of the extraction mode on the yield of bioactive compounds from Crataegus monogyna Jacq. in order to evaluate plant productivity.Methodology - Samples were prepared by extraction of powdered material obtained from top branches, flowers and leaves. Soxhlet extraction, maceration and ultrasound- and microwave-assisted extraction at different experimental conditions were investigated for the exhaustive extraction of hyperoside, vitexin and vitexin-2[prime][prime]-O-rhamnoside. The phytocomponents were identified and quantified by HPLC-UV/PAD, comparing HPLC retention times and UV spectra of individual peaks with those of the standards analysed under the same conditions.Results - An easy-to-use HPLC isocratic method suitable for the quantification of hyperoside, vitexin and vitexin-2[prime][prime]-O-rhamnoside in raw plant extracts was developed. The optimised HPLC methodology was applied to evaluate different extraction procedures. The ultrasound and microwave-assisted extraction protocols showed higher extraction efficiency than the others. In particular, the optimised microwave protocol gave rise to the highest extraction efficiency with high reproducibility.Conclusions - A microwave protocol combined with isocratic HPLC analysis is proposed for the rapid screening of plant materials collected in different environmental conditions in order to evaluate the productivity of Crataegus monogyna Jacq. and to find out the best ecological conditions to cultivate hawthorn in Northern Italy. Copyright © 2008 John Wiley & Sons, Ltd.
A Rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants
Introduction - RNA quality and integrity are critical for many studies in plant molecular biology. High-quality RNA extraction from grapevine and other woody plants is problematic due to the presence of polysaccharides, polyphenolics and other compounds that bind or co-precipitate with the RNA.Objective - To develop an optimised cetyltrimethylammonium bromide (CTAB)-based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from polysaccharide-rich tissues of several plants.Methodology - Several changes were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4°C, the sample weight was decreased and the concentrations of PVP-40 and LiCl were increased reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with six different RNA extraction methods from three grapevine tissues, namely, in vitro plantlets, and leaves and mature canes from actively growing field vines.Results - The rapid CTAB method gave high-quality RNA in only 3 h at low cost with efficiency equal to or higher than that obtained with other time-consuming and expensive protocols. The procedure was applied to RNA extraction from other grapevine tissues and other woody species including olive, lemon, poplar, chestnut, apple, pear, peach, cherry, apricot, plum and kiwi fruit. RNA of high quality could be isolated from all tissues and from all species.Conclusion - The study has shown that the improvement of a CTAB-based protocol allows the rapid isolation of high-quality RNA from grapevine and many woody species. Copyright © 2008 John Wiley & Sons, Ltd.
Safety assessment of food and herbal products containing hepatotoxic pyrrolizidine alkaloids: interlaboratory consistency and the importance of N-oxide determination
Introduction - Two recent mass spectrometry-based reports concerning Senecio scandens yielded remarkably dissimilar pyrrolizidine alkaloid constituents. In both studies, and in a related analysis of Senecio scandens and Tussilago farfara using micellar electrokinetic chromatography, the presence of hazardous N-oxides of the alkaloids was either not considered or was inadequately considered. This raises concerns about the effectiveness of the methodologies used in these, and similar, studies in assessing the pyrrolizidine alkaloid content and the safety of food, food supplements and medicines for human use.Objective - To highlight essential analytical requirements for confident assessment of pyrrolizidine alkaloid-related safety of food and herbal products for human use.Methodology - Direct infusion-ESI MS and HPLC-ESI MS were used to analyse samples derived from liquid-liquid partitioning experiments and from strong cation exchange, solid-phase extraction of pyrrolizidine alkaloids and their N-oxides.Results - A simple solvent partitioning experiment using pure senecionine and senecionine-N-oxide, two constituents reported in one of the mass spectrometry-based studies of S. scandens, clearly demonstrated the inadequacy of the reported method to detect and quantitate hazardous pyrrolizidine alkaloid N-oxide components. A preliminary LCMS analysis of commercially-prepared extracts of comfrey roots (Symphytum officinale and S. uplandicum s. l.) was used as a model to highlight the analytical importance of N-oxides in the safety assessment of pyrrolizidine alkaloid-containing medicinal herbs.Conclusions - This study highlighted significant differences in the reported identification of pyrrolizidine alkaloids from the same plant species, and clearly demonstrated the inadequacy of some procedures to include N-oxides in the assessment of pyrrolizidine alkaloid-related safety of food and herbal products. Copyright © 2008 John Wiley & Sons, Ltd.
Study on the extraction, purification and quantification of jasmonic acid, abscisic acid and indole-3-acetic acid in plants
Introduction - Jasmonic acid (JA), abscisic acid (ABA) and indole-3-acetic acid (IAA) are important plant hormones. Plant hormones are difficult to analyse because they occur in small concentrations and other substances in the plant interfere with their detection.Objective - To develop a new, inexpensive procedure for the rapid extraction and purification of IAA, ABA and JA from various plant species.Methodology - Samples were prepared by extraction of plant tissues with methanol and ethyl acetate. Then the extracts were further purified and enriched with C18 cartridges. The final extracts were derivatised with diazomethane and then measured by GC-MS. The results of the new methodology were compared with those of the Creelman and Mullet procedure.Results - Sequential elution of the assimilates from the C18 cartridges revealed that IAA and ABA eluted in 40% methanol, while JA subsequently eluted in 60% methanol. The new plant hormone extraction and purification procedure produced results that were comparable to those obtained with the Creelman and Mullet's procedure. This new procedure requires only 0.5 g leaf samples to quantify these compounds with high reliability and can simultaneously determine the concentrations of the three plant hormones.Conclusion - A simple, inexpensive method was developed for determining endogenous IAA, ABA and JA concentrations in plant tissue. Copyright © 2008 John Wiley & Sons, Ltd.
Flavonoid glucuronides with anti-leukaemic activity from Polygonum amphibium L.
Introduction - The chemical and pharmaceutical studies carried out on species from Polygonum L. genus showed biological activity both of the extracts and the components isolated from them. These results were the impulse to examine Polygonum amphibium L.Objective - The aim of this study was the isolation of active components from methanol extract and the determination of their cytotoxic effect on human leukaemic cell lines.Methodology - Three flavonoid components from butanol soluble fractions of methanol extract by CC and PC preparative chromatography were isolated. Their structures were established on the basis of 1H, 13C and correlation (DEPT, H-H, COSY, HMQC, HMBC) NMR, UV and FAB-MS spectroscopic techniques. The evaluation of the anti-leukaemic activities of 1 and 2 against Jurkat and HL60 cell lines was carried out in vitro using annexin V fluorescence assay.Results - Two new flavonoid glucuronides, quercetin-3-O-[beta]-glucuronide (1) and quercetin-3-O-[alpha]-rhamnosyl-(1 [rarr] 2)-[beta]-glucuronide (2), and kaempferol-3-O-[alpha]-rhamnosyl-(1 [rarr] 2)-[beta]-glucuronide (3), were isolated from Polygonum amphibium L. It was demonstrated that the glucuronides of quercetin are able to induce apoptosis in the tested human leukaemic cells. These compounds penetrate through cytoplasm to the cellular nucleus of the cultured cells, and give intensive apoptotic responses in the stimulated leukaemic cells. The number of apoptotic cells increased with the concentration (1 nm to 10 µm) of 1 or 2 and periods of exposure (1-3 days).Conclusion - Compounds 1 and 2 may be considered good candidates for leukaemia chemotherapeutic agents. Copyright © 2008 John Wiley & Sons, Ltd.
Preparative isolation of closely-related xanthones from Gentianella amarella ssp. acuta by High-speed countercurrent chromatography
Introduction - A methanolic extract from Gentianella amarella ssp. acuta was shown to contain several xanthones exhibiting acetylcholinesterase inhibitory activity. These xanthones were difficult to separate by conventional LC techniques, which prevented the isolation of pure compounds in sufficient amounts to perform in-depth biological testing.Objective - To develop a suitable preparative method for the separation of closely related xanthones.Methodology - The methanolic extract was first partitioned with solvents of increasing polarity, in order to separate glycosides from xanthone aglycones. High-speed countercurrent chromatography (HSCCC) methods were then optimised for the fractionation of both polar and non-polar extracts.Results - The use of HSCCC enabled the separation of xanthones which co-eluted by HPLC. Ten closely related xanthones - three of which were isomeric - were successfully isolated by developing suitable solvent systems. All compounds were obtained in sufficient amounts to allow further biological assays (e.g. up to 250 mg), including even minor compounds that were not detectable by analytical HPLC.Conclusion - The orthogonality of HSCCC with HPLC and the absence of solid-phase supports enabled the detection, separation and preparative isolation of closely related compounds which were difficult to resolve by other techniques. Copyright © 2008 John Wiley & Sons, Ltd.
Analysis of pharmaceutical samples of Resina Draconis by HPLC-PAD
Introduction: The quality evaluation of traditional Chinese medicine (TCM) represents a particular challenge owing to the complexity of the matrix, which renders separation and identification of the individual components extremely difficult. In recent years, fingerprinting of TCMs has played a dominant role in quality control. Resina Draconis was authorised as a new TCM in 1991, but a satisfactory HPLC fingerprint method for this preparation has not yet been published.Objective: To develop a simple and reliable protocol for the quality control of Resina Draconis using an HPLC-PAD method.Methodology: The TCM was extracted with methanol at room temperature. Chromatography was carried out using a Lichrospher C18 column eluted with a linear gradient of acetonitrile (A) and water containing 0.1% phosphoric acid (B), initially at 30:70 (A:B) and changing to 60:40 in 90 min. UV (PAD) spectra were acquired in the range 210-400 nm.Results: Four chromatograms of samples of Resina Draconis obtained from different pharmaceutical factories showed 20 peaks in common. The average chromatogram was taken as a template from which the correlation coefficients and cosine ratios of the samples were determined. Whereas the contents of individual components in each sample were different, overall the samples were extremely similar one to another, and the products from different pharmaceutical factories were consistent.Conclusion: A reliable and validated HPLC method has been developed for the fingerprint analysis of Resina Draconis that can be applied for the quality control of this TCM. Copyright © 2008 John Wiley & Sons, Ltd.
Quantification of seed oil from species with varying oil content using supercritical fluid extraction
Introduction: The quantity and composition of seed oil affects seed viability and storability and hence the value of a species as a resource for nutrition and plant conservation. Supercritical fluid extraction with carbon dioxide (SFE-CO2) offers a rapid, environmentally friendly alternative to traditional solvent extraction.Objective: To develop a method using SFE-CO2 to quantify the seed oil content in a broad range of species with high to low oil contents.Methodology: Seed oil was extracted using SFE-CO2 from four crop species representing high, medium and low oil content: Helianthus annuus, Asteraceae, with ca. 55% oil; Brassica napus, Brassicaceae, with ca. 50% oil; Glycine max, Fabaceae, with ca. 20% oil; and Pisum sativum, Fabaceae, with ca. 2% oil. Extraction pressures of 5000, 6000 and 7500 psi and temperatures of 40, 60 and 80°C were examined and a second step using 15% ethanol as a modifier included. Oil yields were compared with that achieved from Smalley Butt extraction. The optimised SFE-CO2 method was validated on six species from taxonomically distant families and with varying oil contents: Swietenia humilis (Meliaceae), Stenocereus thurberi (Cactaceae), Sinapis alba (Brassicaceae), Robinia pseudoacacia (Fabaceae), Poa pratensis (Poaceae) and Trachycarpus fortunei (Arecaceae).Results: The two-step extraction at 6000 psi and 80°C produced oil yields equivalent to or higher than Smalley Butt extraction for all species, including challenging species from the Brassicaceae family.Conclusion: SFE-CO2 enables the rapid analysis of seed oils across a broad range of seed oil contents. Copyright © 2008 John Wiley & Sons, Ltd.
Fingerprint of Hedyotis diffusa Willd. by HPLC-MS
A HPLC-MS fingerprint method has been developed based on the consistent chromatographic features of the major chemical constituents among 10 batches of Hedyotis diffusa Willd. Chromatographic separation was conducted on a Hypersil-Keystone Hypurity C18 column using methanol:water:acetic acid as the mobile phase. Major compounds, including oleanolic acid, ursolic acid and ferulic acid, were analysed by HPLC-MS. Their analysis was ascertained by comparison with data derived from the standard compounds. The HPLC-MS fingerprint was successfully applied to analyse and differentiate samples from different geographical origins, or processing methods. H. diffusa was well distinguished from Hedyotis chrysotricha by HPLC-MS. Therefore the establishment of fingerprint of H. diffusa is critical in assessing and controlling its overall quality. Copyright © 2008 John Wiley & Sons, Ltd.
Evaluation of viability assays for anthocyanins in cultured cells
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a widely accepted cytotoxicity assay which can produce inaccurate results due to possible interference with the antioxidant property of anthocyanins. Alternative methods to the MTT assay, such as BrdU (DNA-based) and CellTiter-Glo (ATP-based) assays were evaluated to assess anthocyanin cytotoxicity, derived from blackberry in LNCaP, MCF-7 and MDA-MB-453 cell lines. The standard cell counting method was the reference assay. Greater correlation of cell viability values following anthocyanin exposure was obtained from multiple cell lines with the alternative assays when compared with cell counting. MTT and cell counting results were not always correlated, albeit this was a function of cell type. In particular, poor correlations between cell counting and MTT procedures used to assess cytotoxicity of anthocyanins were observed in the MDA-MB-453 cell lines. Comparison of cytotoxicity derived from alternative assays and the MTT assays with the cell counting method was dependent on the assay procedure and the cell type. The LC50 of blackberry crude extract ranged from 0.4 to 9.4 mg/mL between assays and across all cell lines, whereas a semi-purified anthocyanin extract was not cytotoxic. Cytotoxicity evaluation of polyphenolic-rich extracts using BrdU and CellTiter-Glo assays as alternatives to the MTT method is recommended. Copyright © 2008 John Wiley & Sons, Ltd.
