Antioxidant and Antimicrobial Effects of Baby Leaves of Amaranthus tricolor L. Harvested as Vegetable in Correlation with Their Phytochemical Composition.

Amaranth is used as a spinach replacement; therefore, it is sometimes called Chinese Spinach. So far, the activity of the plant has not been associated with the presence of specific compounds. Three cultivars of Amaranthus tricolor L. were investigated for their antioxidant and antimicrobial activities. The correlation between the bioactivity and metabolite profiles was investigated in order to indicate active compounds in A. tricolor. The phytochemical profile of a total of nine extracts was studied by HPLC-DAD-ESI/HRMS, revealing the presence of 52 compounds. The highest antioxidant activity was noticed in the Red cultivar (0.06 mmol TE/g DE (Trolox Equivalent/Dry Extract Weight) and was related to the presence of amino acids, flavonoids and phenolic acids, as well as individual compounds such as tuberonic acid hexoside. All studied extracts revealed antimicrobial activity. Gram-positive bacteria were more susceptible to N-(carboxyacetyl) phenylalanine, phenylalanine, tuberonic acid and succinic acid and Gram-negative bacteria to dopa, tryptophan, norleucine, tuberonic acid hexoside, quercetin-O-hexoside, luteolin-O-rhamnosylhexoside, luteolin-6-C-hexoside succinic acid, gallic acid-O-hexoside, dihydroxybenzoic acid and hydroxybenzoic acid. Maleic acid showed promising antifungal activity. In summary, A. tricolor is a good source of antioxidant and antimicrobial compounds.

Caprylic acid enhances hydroxyhexylitaconic acid production in Aspergillus niger S17-5.

AIM: Aspergillus niger S17-5 produces two alkylitaconic acids, 9-hydroxyhexylitaconic acid (9-HHIA) and 10-hydroxyhexylitaconic acid (10-HHIA), which have cytotoxic and polymer building block properties. In this study, we characterized the production of 9-HHIA and 10-HHIA by addition of their expected precursor, caprylic acid, to a culture of A. niger S17-5, and demonstrated batch fermentation of 9-HHIA and 10-HHIA in a jar fermenter with DO-stat. METHODS AND RESULTS: Production titres of 9-HHIA and 10-HHIA from 3% glucose in a flask after 25 days cultivation were 0·35 and 1·01 g l-1 respectively. Addition of 0·22 g l-1 of caprylic acid to a suspension of resting cells of A. niger S17-5 led to 32% enhancement of total 9-HHIA and 10-HHIA production compared to no addition. No enhancement of the production of 9-HHIA or 10-HHIA by the addition of oxaloacetic acid was observed. Addition of caprylic acid to the culture at mid-growth phase was more suitable for 9-HHIA and 10-HHIA production due to less cell growth inhibition by caprylic acid. DO-stat batch fermentation with 3% glucose and 14·4 g l-1 of caprylic acid in a 1·5 l jar fermenter resulted in the production titres of 9-HHIA and 10-HHIA being 0·48 and 1·54 g l-1 respectively after 10 days of cultivation. CONCLUSIONS: Addition of caprylic acid to the culture of A. niger S17-5 enhances 9-HHIA and 10-HHIA production. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that 9-HHIA and 10-HHIA are synthesized with octanoyl-CoA derived from caprylic acid, and that the supply of octanoyl-CoA is a rate-limiting step in 9-HHIA and 10-HHIA production. To the best of our knowledge, this is the first report regarding the fermentation of naturally occurring itaconic acid derivatives in a jar fermenter.

Simple Synthesis of 17-ß-O-hemisuccinate of Stanozolol for Immunoanalytical Methods.

The use of doping in sports is a global problem that affects athletes around the world. Among the different methods developed to detect doping agents in biological samples, there are antibody-based methods that need an appropriate hapten design. Steroids with a hydroxyl group can be converted to the corresponding hemisuccinates. A novel approach to the synthesis of 17ß-O-hemisuccinate of the common doping agent stanozolol is described here. Acylation of stanozolol with methyl 4-chloro-4-oxobutyrate/4-dimethylaminopyridine, followed by mild alkaline hydrolysis with methanolic sodium hydroxide at room temperature, gave the simultaneous protection and deprotection of pyrazole-nitrogen atoms. The proposed new synthetic method allows the desired hemisuccinate derivative to be obtained in only two steps, and with a good total yield starting from stanozolol.

Enhanced marine monitoring and toxicity study of oil spill dispersants including Corexit EC9500A in the presence of diluted bitumen.

Observations made for the analysis of the oil spill dispersant tracer dioctyl sulfosuccinate (DOSS) during LC50 toxicity testing, highlighted a stability issue for this tracer compound in seawater. A liquid chromatography high-resolution quadrupole time-of-flight mass spectrometry (LC/QToF) was used to confirm monooctyl sulfosuccinate (MOSS) as the only significant DOSS breakdown product, and not the related isomer, 4-(2-ethylhexyl) 2-sulfobutanedioate. Combined analysis of DOSS and MOSS was shown to be applicable to monitoring of spill dispersants Corexit® EC9500A, Finasol OSR52, Slickgone NS, and Slickgone EW. The unassisted conversion of DOSS to MOSS occurred in all four oil spill dispersants solubilized in seawater, although differences were noted in the rate of MOSS formation. A marine microcosm study of Corexit EC9500A, the formulation most rapid to form MOSS, provided further evidence of the stoichiometric conversion of DOSS to MOSS under conditions relevant to real world dilbit spill. Results supported combined DOSS and MOSS analysis for the monitoring of spill dispersant in a marine environment, with a significant extension of sample collection time by 10 days or longer in cooler conditions. Implications of the unassisted formation of MOSS and combined DOSS:MOSS analysis are discussed in relation to improving dispersant LC50 toxicity studies.

Targeted metabolomics to investigate antimicrobial activity of itaconic acid in marine molluscs.

INTRODUCTION: Itaconic acid (ITA) has recently been identified as an antimicrobial metabolite in mammalian immune cells. The presence of ITA was also reported in different tissues of marine molluscs, indicating its role as an endogenous metabolite of molluscs. In addition, the accumulation of ITA has been observed in different tissues of mussels following pathogen challenges. However, the concentration of ITA in mussel tissues and the possible role of this metabolite in the molluscan innate immune system remain unknown. OBJECTIVES: This study aims to quantitatively measure ITA levels in different tissues of marine mussels following an experimental challenge with Vibrio sp. DO1 isolate, and to identify the antimicrobial role of ITA in the innate immune system through the measurement of metabolic and immune alterations in tissues following the challenge. METHODS: In this study, adult Perna canaliculus mussels were experimentally challenged with a pathogenic Vibrio sp. DO1 isolate. The metabolite profiles of five different tissues, including mantle, gill, muscle, hepatopancreas and haemolymph were obtained, and levels of ITA in each tissue were characterized using a gas chromatography-mass spectrometry (GC-MS) metabolomics approach. Flow cytometry was also employed to measure cell health parameters, including oxidative stress via reactive oxygen species (ROS) production, apoptosis via changes in mitochondrial membrane potential (MMP) and haemocyte viability. RESULTS: The ITA levels in mantle, gill, muscle and hepatopancreas tissues at 18-h post infection (hpi) with Vibrio sp. were 40.31, 41.71, 11.61 and 41.66 ng mg-1, respectively. In haemolymph, the level of ITA was significantly increased from 95.25 ng ml-1 at 6 hpi to 174.36 ng ml-1 at 18 hpi and 572.12 ng ml-1 at 60 hpi. In line with the accumulation of ITA, we observed increased levels of metabolites within the tricarboxylic acid (TCA) cycle, anti-inflammatory metabolites and alterations of other metabolites associated with immune responses of the host. The flow cytometry analyses revealed increases in ROS production, apoptotic cells and decreases in cell viability. CONCLUSIONS: We reported on the production of ITA in different tissues of P. canaliculus mussels challenged with a marine pathogen which confirmed ITA as an antimicrobial metabolite. The findings revealed insights into the biosynthesis of ITA and suggests its role in antimicrobial and anti-inflammatory activities in the innate immune system. This study also provided insights into the innate immune system of bivalves and highlighted the potential use of ITA as a biomarker for shellfish health assessment in aquaculture.

Re-engineering Escherichia coli KJ122 to enhance the utilization of xylose and xylose/glucose mixture for efficient succinate production in mineral salt medium.

Escherichia coli KJ122 was previously engineered to produce high concentration and yield of succinate in mineral salt medium containing glucose and sucrose under anaerobic conditions. However, this strain does not efficiently utilize xylose. To improve the xylose uptake and utilization in the strain KJ122, xylFGH and xylE genes were individually and simultaneously deleted. E. coli KJ12201 (KJ122::ΔxylFGH) exhibited superior abilities in growth, xylose consumption, and succinate production compared to those of the parental strain KJ122. However, E. coli KJ12202 (KJ122::ΔxylE) lessened xylose consumption due to an ATP deficit for metabolizing xylose thus making succinate production from xylose not preferable. Moreover, E. coli KJ12203 (KJ122::ΔxylFGHΔxylE) exhibited an impaired growth on xylose due to lacking of xylose transporters. After performing metabolic evolution, the evolved KJ12201-14T strain exhibited a great improvement in succinate production from pure xylose with higher concentration and productivity about 18 and 21%, respectively, compared to KJ12201 strain. During fed-batch fermentation, KJ12201-14T also produced succinate from xylose at a concentration, yield, and overall productivity of 84.6 ± 0.7 g/L, 0.86 ± 0.01 g/g and 1.01 ± 0.01 g/L/h, respectively. KJ12201 and KJ12201-14T strains co-utilized glucose/xylose mixture without catabolite repression. Both strains produced succinate from glucose/xylose mixture at concentration, yield, and overall and specific productivities of about 85 g/L, 0.85 g/g, 0.70 g/L/h, and 0.44 g/gCDW/h, respectively. Based on our results, KJ12201 and KJ12201-14T strains exhibited a greater performance in succinate production from xylose containing medium than those of other published works. They would be potential strains for the economic bio-based succinate production from xylose.

Metabolic and immunological responses of male and female new Zealand Greenshell™ mussels (Perna canaliculus) infected with Vibrio sp.

Massive mortalities due to pathogens are routinely reported in bivalve cultivation that have significant economic consequences for the global aquaculture industry. However, host-pathogen interactions and infection mechanisms that mediate these interactions are poorly understood. In addition, gender-specific immunological responses have been reported for some species, but the reasons for such differences have not been elucidated. In this study, we used a GC/MS-based metabolomics platform and flow cytometry approach to characterize metabolic and immunological responses in haemolymph of male and female mussels (Perna canaliculus) experimentally infected with Vibrio sp. Sex-based differences in immunological responses were identified, with male mussels displaying higher mortality, oxidative stress and apoptosis after pathogen exposure. However, central metabolic processes appeared to be similar between sexes at 24 h post injection with Vibrio sp. DO1. Significant alterations in relative levels of 37 metabolites were detected between infected and uninfected mussels. These metabolites are involved in major perturbations on the host's innate immune system. In addition, there were alterations of seven metabolites in profiles of mussels sampled on the second day and mussels that survived six days after exposure. These metabolites include itaconic acid, isoleucine, phenylalanine, creatinine, malonic acid, glutaric acid and hydroxyproline. Among these, itaconic acid has the potential to be an important biomarker for Vibrio sp. DO1 infection. These findings provide new insights on the mechanistic relationship between a bivalve host and a pathogenic bacterium and highlight the need to consider host sex as a biological variable in future immunological studies.

Insight into the Chemical Diversity of Late/Ice Harvest Gewürztraminer Wine.

Late harvest (LHW) and ice harvest (IHW) Gewürztraminer wine samples from Croatia (Ilok) were investigated. Their technological parameters, chromaticity coordinates, total phenols content, and antioxidant capacity were determined. 5-(Hydroxymethyl)furfural, xanthine, and trans-caftaric acid were analyzed in the samples by high performance liquid chromatography (HPLC-DAD). Headspace solid-phase microextraction (HS-SPME) followed by gas chromatography and mass spectrometry (GC/MS) analysis revealed isoamyl alcohol as predominant compound (21.25 - 60.30%). Diethyl succinate, 2-phenylethanol, and benzaldehyde were also abundant. Ethyl octanoate (1.48 - 5.70%) and ethyl caprate (0.48 - 4.83%) decreased significantly in LHW, being the lowest in IHW. Two solvents were applied for the samples extraction (solvent A - pentane/diethyl ether 1:2 (v/v) and solvent B - dichloromethane), and the extracts were analyzed by GC/MS. Ethyl hydrogen succinate (solvent A: 27.30 - 52.04%; solvent B: 28.04 - 46.69%) and diethyl succinate (solvent A: 5.21 - 18.2%; solvent B: 2.66 - 7.72%) were predominant in IHW and LHW. Aromatic alcohols were also found: 2-phenylethanol (solvent A: 7.07 - 21.09%; solvent B: 5.50 - 11.82%), 4-hydroxybenzyl alcohol (solvent A: 1.45 - 6.68%; solvent B: 2.47 - 12.16%) and benzyl alcohol (solvent A: 0.10 - 0.77%). The obtained results complement a previous study on IHW (Gewürztraminer) from Croatia providing new features and indicating great chemical diversity among the samples.

Identification and occurrence of the hydroxamate siderophores aerobactin, putrebactin, avaroferrin and ochrobactin C as virulence factors from entomopathogenic bacteria.

Effective iron acquisition and fine-tuned intracellular iron storage systems are the main prerequisites for a successful host invasion by a pathogen. Bacteria have developed several different strategies to sequester this essential element from their environment, one relies on the secretion of low molecular weight compounds with high affinity for ferric iron, the so-called siderophores. Here, we report hydroxamate siderophore structures produced by entomopathogenic bacteria of the species Xenorhabdus and Photorhabdus, which are known for their potential to produce bioactive natural products, required for their role as nematode symbiont and insect pathogen. Four siderophores could be identified, namely aerobactin, putrebactin, avaroferrin and ochrobactin C, which was found previously only in marine bacteria. While the putrebactin and avaroferrin producing biosynthesis gene cluster (BGC) is more widespread and most likely was present in a common ancestor of these bacteria, the aerobactin and ochrobactin producing BGC was probably taken up by a few strains individually. For aerobactin a role in virulence towards Galleria mellonella larvae is shown.

Enhanced itaconic acid production by self-assembly of two biosynthetic enzymes in Escherichia coli.

Here, we described a novel strategy for the production of itaconic acid in Escherichia coli by self-assembly of aconitase (ACO) and cis-aconitate decarboxylase (CAD) existing in the metabolic pathway of itaconic acid via the protein-peptide interactions of PDZ domain and PDZ ligand. Co-expression of ACO and CAD in E. coli (uCA) resulted in low levels of itaconate (117.25 mg/L) after 48 h fermentation while the itaconate titre was significantly improved up to 222.15 mg/L by self-assembly of ACO-PDZ (APd) and CAD-PDZlig (CPl) in E. coli (sPP) under the same conditions. To further confirm the effect of self-assembly, the itaconate catalyzed from sodium citrate was determined. The sPP was extra efficacious in the early catalytic period, showing approximately threefold itaconate yields increased after 2 h catalysis, when compared to uCA. Furthermore, the itaconate production of sPP was increased from 5 to 8.7 g/L after 30 h of reaction compared to uCA. This self-assembly strategy showed remarkable potential for the further improvement of itaconate production. Biotechnol. Bioeng. 2017;114: 457-462. © 2016 Wiley Periodicals, Inc.

Process development of itaconic acid production by a natural wild type strain of Aspergillus terreus to reach industrially relevant final titers.

Itaconic acid is a promising organic acid and is commercially produced by submerged fermentation of Aspergillus terreus. The cultivation process of the sensitive filamentous fungus has been studied intensively since 1932, with respect to fermentation media components, oxygen supply, shearing rate, pH value, or culture method. Whereas increased final titers were achieved over the years, the productivity has so far remained quite low. In this study, the impact of the pH on the itaconic acid production was investigated in detail. The pH during the growth and production phase had a significant influence on the final itaconic acid concentration and pellet diameter. The highest itaconic acid concentration of 160 g/L was achieved at a 1.5-L scale within 6.7 days by raising and controlling the pH value to pH 3.4 in the production phase. An ammonia solution and an increased phosphate concentration were used with an itaconic acid yield of 0.46 (w/w) and an overall productivity of 0.99 g/L/h in a fed-batch mode. A cultivation with a lower phosphate concentration resulted in an equal final concentration with an increased yield of 0.58 (w/w) after 11.8 days and an overall productivity of 0.57 g/L/h. This optimized process was successfully transferred from a 1.5-L scale to a 15-L scale. After 9.7 days, comparable pellet morphology and a final concentration of 150 g/L itaconic acid was reached. This paper provides a process strategy to yield a final titer of itaconic acid from a wild-type strain of A. terreus which is in the same range as the well-known citric acid production.

Targeted Metabolic Profiling of the Tg197 Mouse Model Reveals Itaconic Acid as a Marker of Rheumatoid Arthritis.

Rheumatoid arthritis is a progressive, highly debilitating disease where early diagnosis, enabling rapid clinical intervention, would provide obvious benefits to patients, healthcare systems, and society. Novel biomarkers that enable noninvasive early diagnosis of the onset and progression of the disease provide one route to achieving this goal. Here a metabolic profiling method has been applied to investigate disease development in the Tg197 arthritis mouse model. Hind limb extract profiling demonstrated clear differences in metabolic phenotypes between control (wild type) and Tg197 transgenic mice and highlighted raised concentrations of itaconic acid as a potential marker of the disease. These changes in itaconic acid concentrations were moderated or indeed reversed when the Tg197 mice were treated with the anti-hTNF biologic infliximab (10 mg/kg twice weekly for 6 weeks). Further in vitro studies on synovial fibroblasts obtained from healthy wild-type, arthritic Tg197, and infliximab-treated Tg197 transgenic mice confirmed the association of itaconic acid with rheumatoid arthritis and disease-moderating drug effects. Preliminary indications of the potential value of itaconic acid as a translational biomarker were obtained when studies on K4IM human fibroblasts treated with hTNF showed an increase in the concentrations of this metabolite.

Itaconic acid production from glycerol using Escherichia coli harboring a random synonymous codon-substituted 5'-coding region variant of the cadA gene.

Aspergillus terreus cadA, encoding cis-aconitate decarboxylase, is an essential gene for itaconic acid (IA) biosynthesis, but it is primarily expressed as insoluble aggregates in most industrial hosts. This has been a hurdle for the development of recombinant strategies for IA production. Here, we created a library of synonymous codon variants (scv) of the cadA gene containing synonymous codons in the first 10 codons (except ATG) and screened it in Escherichia coli. Among positive clones, E. coli scvCadA_No8 showed more than 95% of expressed CadA in the soluble fraction, and in production runs, produced threefold more IA than wild-type E. coli in Luria-Bertani broth supplemented with 0.5% glucose. In M9 minimal media containing 0.85 g/L citrate and 1% glycerol, E. coli scvCadA_No8 produced 985.6 ± 33.4 mg/L IA during a 72-h culture after induction with isopropyl ß-D-1-thiogalactopyranoside. In a 2-L fed-batch fermentation consisting of two stages (growth and nitrogen limitation conditions), we obtained 7.2 g/L IA by using E. coli by introducing only the scv_cadA gene and optimizing culture conditions for IA production. These results could be combined with metabolic engineering and generate an E. coli strain as an industrial IA producer. Biotechnol. Bioeng. 2016;113: 1504-1510. © 2015 Wiley Periodicals, Inc.

Quantitative and qualitative evaluation of kernel anthocyanins from southwestern United States blue corn.

BACKGROUND: Anthocyanin-rich blue corn is an emerging specialty crop in the USA. The antioxidant properties of blue corn offer health benefits in the human diet. The objectives of this study were to identify, characterize and quantify the anthocyanins from blue corn. Hypotheses tested were that total anthocyanin content was similar among southwestern US accessions and that it would vary across locations. It was also examined whether different anthocyanin components were unique to certain genotypes. RESULTS: Across all locations and accessions, an average of 0.43 g kg(-1) total anthocyanin content (TAC) was observed. Accessions Santa Clara Blue and Ohio Blue displayed the highest TAC. The TAC of accession Flor del Rio was lower by nearly a factor of six. A total of five anthocyanin components were identified. Cyanidin 3-glucoside was the most abundant, followed by pelargonidin and peonidin 3-glucoside. Succinyl and disuccinyl glycosidic forms of cyanidin were also identified. Cyanidin 3-disuccinylglucoside was newly identified as a novel form of anthocyanin. CONCLUSION: Quantitative and qualitative anthocyanin expression was determined to be relatively stable across multiple southwestern environments. Increased expression of red and purple pigmentation in accession Flor del Rio appeared to be associated more with reduced TAC and cyanidin 3-glucoside than with elevated pelargonidin per se. A previously unreported anthocyanin component in blue corn, cyanidin 3-disuccinylglucoside, is present in southwestern landraces. © 2016 Society of Chemical Industry.

A deficiency of manganese ions in the presence of high sugar concentrations is the critical parameter for achieving high yields of itaconic acid by Aspergillus terreus.

Itaconic acid (IA), an unsaturated dicarboxylic acid with a high potential as a platform for chemicals derived from sugars, is industrially produced by large-scale submerged fermentation by Aspergillus terreus. Although the biochemical pathway and the physiology leading to IA is almost the same as that leading to citric acid production in Aspergillus niger, published data for the volumetric (g L(-1)) and the specific yield (mol/mol carbon source) of IA are significantly lower than for citric acid. Citric acid is known to accumulate to high levels only when a number of nutritional parameters are carefully adjusted, of which the concentration of the carbon source and that of manganese ions in the medium are particularly important. We have therefore investigated whether a variation in these two parameters may enhance IA production and yield by A. terreus. We show that manganese ion concentrations < 3 ppb are necessary to obtain highest yields. Highest yields were also dependent on the concentration of the carbon source (D-glucose), and highest yields (0.9) were only obtained at concentrations of 12-20 % (w/v), thus allowing the accumulation of up to 130 g L(-1) IA. These findings perfectly mirror those obtained when these parameters are varied in citric acid production by A. niger, thus showing that the physiology of both processes is widely identical. Consequently, applying the fermentation technology established for citric acid production by A. niger citric acid production to A. terreus should lead to high yields of IA, too.

[Simultaneous determination of seven components in Pudilan Xiaoyan oral liquid by HPLC].

OBJECTIVE: To establish an HPLC method for the content determination of baicalin, wogonin, chlorogenic acid, caffeic acid, cichoric acid, corynoline and adenosine in Pudilan Xiaoyan oral liquid. METHOD: The analysis was performed on a Phenomenex Luna C18 column (4.6 mm x 250 mm, 5 µm) with a gradient mobile phase of methanol-0.1% trifluoroacetic acid solution system at flow rate of 1.0 mL · min(-1). The detective wavelength was at 280 nm. The column temperature was 30 °C. RESULT: The standard curves of seven studied components show good linearity in their concentration ranges with r ≥ 0.999 6. The average recovery was 98.73%-102.1% with RSD less than 2.6%. CONCLUSION: The method is rapid, simple and accurate, and can be applied for the quality control of Pudilan Xiaoyan oral liquid.

Synthesis of anaerobic degradation biomarkers alkyl-, aryl- and cycloalkylsuccinic acids and their mass spectral characteristics.

Anaerobic biodegradation of petroleum hydrocarbons has been reported to proceed predominantly via fumarate addition to yield substituted succinate metabolites. These metabolites, commonly regarded as signature biomarkers, are specific indicators of anaero- bic hydrocarbon degradation by microbial activity. To the best of our knowledge, mass spectrometry information for 2-(1-methylalkylj succinic acids, 2-arylsuccinic acids, 2-cycloalkylsuccinic acids and/or their derivatives is still incomplete, especially for the analysis of environmental samples. Here, a novel approach is proposed for the successful synthesis of five hydrocarbon-derived succinic acids. The characteristic fragments of 2-[1-methylalkyllsuccinic acid diesters were investigated by four derivatization processes (methyl, ethyl, n-butyl and trimethylsilyl esterification], some of which are not available in official Libraries. Under electron ionization mass spec- trometry conditions, informative fragments of various molecular masses have been obtained. Results confirmed characteristic differ- ences among the derivatization processes of the chemically synthesized compounds. In the case of 2-[cyclo)alkylsuccinate esters, four intermediate fragments were observed at m/z 114 + 14n, 118 + 28n, [M - [17 + 14n1]]+ and [M - (59 + 14n)]+ (n = 1, 2 and 4 for methyl, ethyl and n-butyl ester]. However, for silylation the abundant fragment ions are at m/z 262, 217, 172, 147, 73 and [M - 15]+. These data provide information for the identification of hydrocarbon-derived succinic acids as anaerobic biodegradation intermediates in hydrocarbons- rich environments.

Quantification of phenylpropanoids in commercial Echinacea products using TLC with video densitometry as detection technique and ANN for data modelling.

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

[Determination of dactylorhin A and militarine in three varieties of Cremastrae Pseudobulbus/Pleiones Pseudobulbus by HPLC].

To establish an HPLC method for determination of dactylorhin A and militarine in Cremastrae Pseudobulbus/Pleiones Pseudobulbus. The analysis was achieved on an Alltech Prevail C18 column (4. 6 mm x 250 mm, 5 microm) using a mobile phase of acetonitrile (A), water (B) gradient elution in a total run time of 35 min (0 min, 20:80; 30 min, 55:45; 35 min, 55:45) and a diode array detector was set at 224 nm. The flow rate was 0.8 mL x min(-1). The assay displayed good linearity over the concentration range of 0.257-9.95 microg (r = 0.999 8), and 0.128-10.27 microg (r = 0.999 9), respectively. The average recoveries (n = 9) were 94.70% and 102.8% for dactylorhin A and militarine, respectively. The method is accurate, quick, simple and reproducibility. It can be used for the quality control of Pleione bulbocodioides and Pleione yunnanensis.

[Simultaneous determination of five organic acids in Kudiezi injection by HPLC].

The aim was to develop a high performance liquid chromatography method for simultaneous determination of five organic acids in Kudiezi injection. The Diamonsil C18 column (4.6 mm x 200 mm, 5 microm) was adopted with acetonitrile and water as the mobile phase at a gradient mode program. The flow rate was 1.0 mL min-1 , detection wavelength was 325 nm, and column temperature was 35 degree C. The linear range of monocaffeyltartaric acid, chlorogenic acid, caffeic acid, ferulic acid, and chicoric acid were 0. 64-81.60 (r =0. 999 9),0.09-11. 10 (r =0.999 8) ,0.09-11.30 (r =0. 999 8),0.10-12.80 (r =0.999 9),0.43-55. 50 mg L-1 (r = 0.999 8) , respectively. The average recoveries were 101.8% ,100. 9% ,99. 24% ,99. 83% ,101.9%, respectively, with RSD of less than 2.0%. The developed HPLC method was simple, sensitive and accurate with good repeatability. This work provided helpful information for comprehensive quality control of Kudiezi injection. [Key words] Kudiezi injection; organic acids; content determination; HPLC