Search

This study employed a diverse approach to extract antioxidant peptides from red seaweed Palmaria palmata , recognized for its comparatively high protein content. Initially, an aqueous extraction of the entire seaweed was performed, followed by enzymatic hydrolysis of the solid residues prepared from the first step. The effects of three different pH levels (3, 6, and 9) during the aqueous extraction were also examined. Results indicated that the solid fraction from the sequential extraction process contained significantly higher levels of proteins and amino acids than other fractions ( p < 0.05). Furthermore, the solid fractions (IC 50 ranging from 2.29 to 8.15 mg.mL -1 ) demonstrated significantly greater free radical scavengers than the liquid fractions (IC 50 ranging from 9.03 to 10.41 mg.mL -1 or not obtained at the highest concentration tested) at both stages of extraction ( p < 0.05). Among the solid fractions, those produced fractions under alkaline conditions were less effective in radical scavenging than the produced fractions under acidic or neutral conditions. The fractions with most effective metal ion chelating activity were the solid fractions from the enzymatic stage, particularly at pH 3 (IC 50 = 0.63 ± 0.04 mg.mL -1 ) and pH 6 (IC 50 = 0.89 ± 0.07 mg.mL -1 ), which were significantly more effective than those from the initial extraction stage ( p < 0.05). Despite no significant difference in the total phenolic content between these solid fractions and their corresponding liquid fractions (3.79 ± 0.05 vs. 3.48 ± 0.02 mg.mL -1 at pH 3 and 2.43 ± 0.22 vs. 2.51 ± 0.00 mg.mL -1 at pH 6) ( p > 0.05), the observed antioxidant properties may be attributed to bioactive amino acids such as histidine, glutamic acid, aspartic acid, tyrosine, and methionine, either as free amino acids or within proteins and peptides.

Marine rhodophyta are known to synthesize specific secondary metabolites, mycosporine-like amino acids (MAAs), to protect themselves from harmful UV-radiation. Shinorine and porphyra-334 are among the most abundant representatives of this compound class. In the present work, a novel approach for their isolation is described. As a first step, a fast centrifugal partition chromatography method, with an aqueous two-phase system comprising water, ethanol, ammonium sulfate and methanol in ascending mode, was developed to isolate the two MAAs from crude aqueous-methanolic extracts of three algal species within 90 min. The compounds could be isolated when just one of them was present in a sample or also both at the same time. By employing solid phase extraction as a second purification step, the individual MAAs were obtained in high purity and good quantity within a much shorter time frame than the established purification protocols, e.g., semi-preparative HPLC. For example, from 4 g Porphyra sp. (Nori) crude extract, 15.7 mg shinorine and 36.2 mg porphyra-334 were isolated. Both were highly pure, as confirmed by TLC, HPLC-MS and NMR analyses.

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus , and 432 proteins by Lentinula edodes . Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.

Protein and amino acids in pollen are important nutritional components for larval development in several insect species, especially in Apoidea. The Bradford assay is a widely used method to measure relative protein content of pollen, which can shed light on pollen quality and consequences to fitness. Prior to using the Bradford assay, protein must be released from pollen grains, often using a mixture of chemical and mechanical fracturation methods. In this study, we tested the efficacy of protein extraction without using mechanical fracturation. We used pollen collected by the solitary bee Osmia lignaria Say to compare two known buffers associated with pollen protein analysis: phosphate-buffered saline and sodium hydroxide, and deionized water, and with different pollen weights from which we quantified protein using the Bradford assay. While all buffers and deionized water were useful in releasing protein from pollen grains collected by O. lignaria, the use of sodium hydroxide resulted in significantly higher protein quantification across all pollen weights. This methodological study can inform future studies of pollen nutrition in pollen-foraging species., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.)

This study presents the validation of a high-performance liquid chromatography diode array detector (HPLC-DAD) method for the determination of different mycosporine-like amino acids (MAAs) in the red alga Bostrychia scorpioides . The investigated MAAs, named bostrychines, have only been found in this specific species so far. The developed HPLC-DAD method was successfully applied for the quantification of the major MAAs in Bostrychia scorpioides extracts, collected from four different countries in Europe showing only minor differences between the investigated samples. In the past, several Bostrychia spp. have been reported to include cryptic species, and in some cases such as B. calliptera , B. simpliciuscula , and B. moritziana , the polyphyly was supported by differences in their MAA composition. The uniformity in the MAA composition of the investigated B. scorpioides samples is in agreement with the reported monophyly of this Bostrychia sp.

Microalgae are known to be rich in protein. In this study, we aim to investigate methods of producing and purifying proteins of 98 microalgae including Chlorella vulgaris, Arthrospira platensis , Nostoc sp., Dunaliella salina , and Pleurochrysis carterae (Baltic Sea). Therefore, we studied their amino acid composition and developed a two-stage protein concentrate purification method from the microalgae biomass. After an additional stage of purification, the mass fraction of protein substances with a molecular weight greater than 50 kDa in the protein concentrate isolated from the biomass of the microalga Dunaliella salina increased by 2.58 times as compared with the mass fraction before filtration. In the protein concentrate isolated from the biomass of the microalga Pleurochrysis cartera , the relative content of the fraction with a molecular weight greater than 50.0 kDa reached 82.4%, which was 2.43 times higher than the relative content of the same fractions in the protein concentrate isolated from this culture before the two-stage purification. The possibilities of large-scale industrial production of microalgae biomass and an expanded range of uses determine the need to search for highly productive protein strains of microalgae and to optimize the conditions for isolating amino acids from them.

The aim of this study was to establish a method for geographical origins identification of Panax notoginseng (P. notoginseng) based on abundant chromatographic spectral information. Characteristic fingerprints of P. notoginseng extracts samples were generated by Multi-wavelength Fusion Profiling (MWFP) method based on the HPLC fingerprints established at three wavelengths of 203 nm, 270 nm and 325 nm. The samples grouping results calculated with the averagely linear quantified fingerprint method (ALQFM) and the unsupervised statistical methods based on fusion fingerprints matches with the geographical origins. The Multi-wavelength Fusion Profiling (MWFP) method has been successfully applied to identification of geographical origins of P. notoginseng and shows the advantages compared with single-channel fingerprints. In addition, eight physiologically active components, including four saponins, two flavones and two amino acids, were identified from the most relevant ingredients of P. notoginseng geographical origins by fusion fingerprint-efficacy relationship analysis. Besides the recognized active saponins, other categories of active ingredients such as flavonoids and amino acids should be paid attention to in the producing areas identification or the quality judgment of P. notoginseng.

This study was designed to profile the metabolites of Isochrysis galbana , an indigenous and less explored microalgae species. 1 H Nuclear Magnetic Resonance (NMR) spectroscopy and Liquid Chromatography-Mass Spectrometry (LCMS) were used to establish the metabolite profiles of five different extracts of this microalga, which are hexane (Hex), ethyl acetate (EtOAc), absolute ethanol (EtOH), EtOH:water 1:1 (AqE), and 100% water (Aq). Partial least square discriminant analysis (PLS-DA) of the generated profiles revealed that EtOAc and Aq extracts contain a diverse range of metabolites as compared to the other extracts with a total of twenty-one metabolites, comprising carotenoids, polyunsaturated fatty acids, and amino acids, that were putatively identified from the NMR spectra. Meanwhile, thirty-two metabolites were successfully annotated from the LCMS/MS data, ten of which (palmitic acid, oleic acid, α-linolenic acid, arachidic acid, cholesterol, DHA, DPA, fucoxanthin, astaxanthin, and pheophytin) were similar to those present in the NMR profile. Another eleven glycerophospholipids were discovered using MS/MS-based molecular network (MN) platform. The results of this study, besides providing a better understanding of I. galbana 's chemical make-up, will be of importance in exploring this species potential as a feed ingredient in the aquaculture industry.

The recovery of amino acids and other important bioactive compounds from the comb penshell ( Atrina pectinata ) using subcritical water hydrolysis was performed. A wide range of extraction temperatures from 140 to 290 °C was used to evaluate the release of proteins and amino acids. The amount of crude protein was the highest (36.14 ± 1.39 mg bovine serum albumin/g) at 200 °C, whereas a further increase in temperature showed the degradation of the crude protein content. The highest amount of amino acids (74.80 mg/g) was at 230 °C, indicating that the temperature range of 170-230 °C is suitable for the extraction of protein-rich compounds using subcritical water hydrolysis. Molecular weights of the peptides obtained from comb penshell viscera decreased with the increasing temperature. SDS-PAGE revealed that the molecular weight of peptides present in the hydrolysates above the 200 °C extraction temperature was ≤ 1000 Da. Radical scavenging activities were analyzed to evaluate the antioxidant activities of the hydrolysates. A. pectinata hydrolysates also showed a particularly good antihypertensive activity, proving that this raw material can be an effective source of amino acids and marine bioactive peptides.

Supercritical fluid chromatography (SFC) has unique separative characteristics distinguished from those of HPLC and gas chromatography. At present, SFC is widely used and there are many applications in various biological, medical, and pharmaceutical fields. In this review, we focus on recently developed novel techniques related to SFC separation including: new column stationary phases, microfluidics, two-dimensional separation, and gas-liquid separation. In addition, we discuss the application of SFC using a water-containing modifier to biological molecules such as amino acids, peptides, and small proteins that had been challenging analytes.

Fast enantiomeric separation of amino acids was studied by liquid chromatography/mass spectrometry (LC/MS) on a chiral crown ether stationary phase. A chiral crown ether bonded silica column (3 mm internal diameter (i.d.), 5 cm long) packed with 3 μm particles was employed instead of a 15 cm column packed with 5 μm particles used in our previous study. In addition, the extra-column variance, becoming more serious for smaller columns, was reduced by replacing 0.127 mm i.d. post-column tubes with shorter, smaller-diameter (0.0635 mm i.d.) tubes. The results demonstrated the benefits of using shorter columns packed with smaller particles and the reduction of the extra-column band broadening for fast enantiomeric separation. Finally, the enantiomeric separation of 18 pairs of proteinogenic amino acids was achieved within 2 min with a resolution (Rs) > 1.5 for each pair using an isocratic mobile phase of acetonitrile/water/trifluoroacetic acid (ACN/W/TFA) = 96/4/0.5, and a flow rate 1.2 mL/min at 30°C. This is the highest throughput method for simultaneous chiral separation of all proteinogenic amino acids except proline to date., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Methamphetamine (MA) is a highly addictive central nervous system stimulant. Drug addiction is not a static condition but rather a chronically relapsing disorder. Hair is a valuable and stable specimen for chronic toxicological monitoring as it retains toxicants and metabolites. The primary focus of this study was to discover the metabolic effects encompassing diverse pathological symptoms of MA addiction. Therefore, metabolic alterations were investigated in human hair following heavy MA abuse using both targeted and untargeted mass spectrometry and through integrated network analysis. The statistical analyses ( t -test, variable importance on projection score, and receiver-operator characteristic curve) demonstrated that 32 metabolites (in targeted metabolomics) as well as 417 and 224 ion features (in positive and negative ionization modes of untargeted metabolomics, respectively) were critically dysregulated. The network analysis showed that the biosynthesis or metabolism of lipids, such as glycosphingolipids, sphingolipids, glycerophospholipids, and ether lipids, as well as the metabolism of amino acids (glycine, serine and threonine; cysteine and methionine) is affected by heavy MA abuse. These findings reveal crucial metabolic effects caused by MA addiction, with emphasis on the value of human hair as a diagnostic specimen for determining drug addiction, and will aid in identifying robust diagnostic markers and therapeutic targets.

The young leaves of green tea become lighter in color than usual when protected from sunlight by a shading net for about two weeks while growing. These leaves are called "shaded white leaf tea" or SWLT. In the eluate of SWLT, the amount of amino acids (361 mg/L) was significantly higher than that in regular tea (53.5 mg/L). Since theanine and arginine, the first and second most abundant amino acids in SWLT, have significant antistress effects, we examined the antistress effect of SWLT on humans. SWLT or placebo green tea (3 g) was eluted with room-temperature water (500 mL). Participants consumed the tea for one week prior to pharmacy practice and continued for 10 days in the practice period. The state-trait anxiety inventory, an anxiety questionnaire, tended to be scored lower in the SWLT group than the placebo, but other stress markers showed no differences. The effect of the difference in SWLT components examined with mice showed that aspartic acid and asparagine, which are abundant in SWLT, counteracted the antistress effects of theanine and arginine. Large amounts of caffeine also interfered with SWLT's antistress effect. Thus, SWLT, which is high in caffeine and amino acids, suppressed depressant behavior in mice.

Nile perch wastewater was biodegraded using two Bacillus species to recover bioactive substances to enhance its reutilization value. The two Bacillus species successfully produced low-molecular-weight substances with a 47.8% degree of hydrolysis. The antioxidant activities of the Nile perch wastewater increased as the biodegradation proceeded, and the culture supernatant exhibited the highest DPPH (80.1%), ABTS (93.1%) and Fe 2+ chelating (88.5%) antioxidant activities at 60 h. The antioxidant potential of the biodegraded Nile perch wastewater was found to be higher than those of other fish hydrolysates. Moreover, the biodegraded Nile perch wastewater exhibited effective antimicrobial activity against Vibrio vulnificus, exhibiting a minimal inhibitory concentration of 585 μg mL -1 . Two-dimensional thin layer chromatography analysis revealed the specific amino acids responsible for the antioxidant activity, and molecular-weight cut-off ultrafiltration revealed that the <2-kDa fraction exhibited the highest antioxidant activity with the lowest IC 50 values (0.43 and 0.22 mg mL -1 for DPPH and ABTS antioxidant activities, respectively). This is the first report of the reutilization of Nile perch wastewater as a natural antioxidant and antimicrobial ingredient for nutraceuticals., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

UV-absorbing compounds, such as mycosporine-like amino acids (MAAs), are a group of secondary metabolites present in many marine species, including red seaweeds. In these organisms, the content and proportion of the composition of MAAs vary, depending on the species and several environmental factors. Its high cosmetic interest calls for research on the content and composition of MAAs, as well as the dynamics of MAAs accumulation in seaweeds from different latitudes. Therefore, this study aimed to survey the content of UV-absorbing MAAs in three Subantarctic red seaweeds during a seasonal cycle. Using spectrophotometric and HPLC techniques, the content and composition of MAAs of intertidal Iridaea tuberculosa , Nothogenia fastigiate , and Corallina officinalis were assessed. Some samples were also analyzed using high-resolution mass spectrometry coupled with HPLC-ESI-MS in order to identify more precisely the MAA composition. I. tuberculosa exhibited the highest MAA values (above 1 mg g -1 of dried mass weight), while C. officinalis showed values not exceeding 0.4 mg g -1 . Porphyra-334 was the main component in N. fastigiata , whereas I. tuberculosa and C. officinalis exhibited a high content of palythine. Both content and composition of MAAs varied seasonally, with high concentration recorded in different seasons, depending on the species, i.e., winter ( I. tuberculosa ), spring ( N. fastigiata ), and summer ( C. officinalis ). HPLC-ESI-MS allowed us to identify seven different MAAs. Two were recorded for the first time in seaweeds from Subantarctic areas (mycosporine-glutamic acid and palythine-serine), and we also recorded an eighth UV-absorbing compound which remains unidentified., Competing Interests: The authors declare no conflict of interest.

The crude drug ysypó hû ( Adenocalymma marginatum DC., Bignoniaceae) is used traditionally by the Guarani of Eastern Paraguayan as a male sexual enhancer. The aim of the present study was to identify the main constituents of the crude drug and to evaluate the in vitro inhibitory activity towards the enzyme phosphodiesterase-5 (PDE-5). The main compounds were isolated by counter-current chromatography (CCC). The metabolites were identified by spectroscopic and spectrometric means. The chemical profiling of the extracts was assessed by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS). The crude extract and main isolated compounds were tested for their PDE-5 inhibitory activity using commercial kits. The iridoid theviridoside and 4-hydroxy-1-methylproline were isolated as the main constituent of the crude drug. Four chlortheviridoside hexoside derivatives were detected for the first time as natural products. Chemical profiling by HPLC-MS/MS led to the tentative identification of nine iridoids, six phenolics, and five amino acids. The crude extracts and main compounds were inactive towards PDE-5 at concentrations up to 500 µg/mL. Iridoids and amino acid derivatives were the main compounds occurring in the Paraguayan crude drug. The potential of ysypó hû as a male sexual enhancer cannot be discarded, since other mechanisms may be involved.

Mycosporine-like amino acids (MAAs) have gained considerable attention as highly active photoprotective candidates for human sunscreens. However, more studies are necessary to evaluate the extraction efficiency of these metabolites in solvents compatible with cosmetics and their subsequent analysis by HPLC. In the present study, MAA extraction using distilled water and 20% methanol in four Rhodophyta was investigated. Different re-dissolution solvents and a C8 and C18 columns were tested for the HPLC analysis. Porphyra-334, shinorine, palythine, palythine-serine, asterina-330, and palythinol were identified by HPLC/ESI-MS.These MAAs were better isolated with the C8 column and using methanol as re-dissolution solvents. Regarding total MAAs concentrations, no differences between the two solvents were found. The highest values were observed injecting them directly in the HPLC. According to these results, distilled water could be an excellent extraction solvent in the production of MAAs extracts and quantification of their total concentrations for different uses in the industry. Nevertheless, the extraction of MAAs using 20% methanol and re-dissolution in pure methanol after dryness is the best option to characterize and identify the most common MAAs in these red algae. Our results entail important implications regarding the use of red macroalgae as promising candidates as environment-friendly sources of natural sunscreens.

Gut microbiota has been implicated in major diseases affecting the human population and has also been linked to triglycerides and high-density lipoprotein levels in the circulation. Recent development in metabolomics allows classifying the lipoprotein particles into more details. Here, we examine the impact of gut microbiota on circulating metabolites measured by Nuclear Magnetic Resonance technology in 2309 individuals from the Rotterdam Study and the LifeLines-DEEP cohort. We assess the relationship between gut microbiota and metabolites by linear regression analysis while adjusting for age, sex, body-mass index, technical covariates, medication use, and multiple testing. We report an association of 32 microbial families and genera with very-low-density and high-density subfractions, serum lipid measures, glycolysis-related metabolites, ketone bodies, amino acids, and acute-phase reaction markers. These observations provide insights into the role of microbiota in host metabolism and support the potential of gut microbiota as a target for therapeutic and preventive interventions.

As part of our efforts to develop a new method for chiral resolution of amino acids with sonochemically modified proteins, we present result that indicates how ovalbumin microspheres (OAMS) interact specifically with l-amino acids from a racemate in solution, leaving an excess of d-enantiomer in the permeate solution. Among different amino acids that interacted with the OAMS, tryptophan (Trp) was the most successfully resolved with 65% enantiomeric excess. A control experiment with native ovalbumin in solution did not show any chiral resolution of amino acids. Interestingly, when the OAMS were pretreated with racemic lysine (Lys) solution and then used for resolution of tryptophan the enantiomeric enrichment of d-tryptophan was raised to 98%. This unanticipated positive effect is discussed in terms of the structural correlation between Trp and Lys, which is less apparent in other amino acids such as phenylalanine., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Quantum dots (QDs) are very attractive nanomaterials for analytical chemistry, due to high photostability, large surface area featuring numerous ways of bioconjugation with biomolecules, usually high quantum yield and long decay times. Their broad absorption spectra and narrow, sharp emission spectra of size-tunable fluorescence make them ideal tools for pattern-based sensing. However, almost always they are applied for specific sensing with zero-dimensional (0D) signal reporting (only peak heights or peak shifts are considered), without taking advantage of greater amount of information hidden in 1D signal (emission spectra), or huge amount of information hidden in 2D fluorescence maps (Excitation-Emission Matrixes, EEMs). Therefore, in this work we propose opposite strategy-non-specific interactions of QDs, which are usually avoided and regarded as their disadvantage, were exploited here for 2D fluorescence fingerprinting. Analyte-specific multivariate fluorescence response of QDs is decoded with the use of Partial Least Squares-Discriminant Analysis. Even though only one type of QDs is studied, the proposed pattern-based method enables to obtain satisfactory accuracy for all studied compounds-various neurotransmitters, amino-acids and oligopeptides. This is a proof of principle of the possibility of the identification of various bioanalytes by such fluorescence fingerprinting with the use of QDs.

The present study sought to evaluate the nutritional composition and physicochemical properties of two dried commercially interesting edible red seaweeds, Gracilaria corticata and G. edulis . Proximate composition of the dried seaweeds revealed a higher content in carbohydrates (8.30 g/100 g), total crude protein (22.84 g/100 g) and lipid content (7.07 g/100 g) in G. corticata than in G. edulis . Fatty acids profile showed that G. corticata samples contain higher concentrations of saturated fatty acids, such as palmitic and stearic acids, and polyunsaturated ones such as α-linolenic and docosahexaenoic acids. Contrariwise, G. edulis contained higher amounts of monounsaturated oleic acid. Total amino acid content was 76.60 mg/g in G. corticata and 65.42 mg/g in G. edulis , being the essential amino acid content higher in G. edulis (35.55 mg/g) than in G. corticata (22.76 mg/g). Chlorophyll a was found in significantly higher amounts in G. edulis (17.14 μg/g) than G. corticata , whereas carotenoid content was significantly higher in G. corticata (12.98 μg/g) than in G. edulis . With respect to physical properties, both water- and oil-holding capacities were similar in both seaweeds, whereas swelling capacity was higher in G. edulis . In view of the results, the present study suggests that G. corticata and G. edulis contains important nutrients for human health and are possible natural functional foods.

This work describes the direct coupling of the in-tube solid-phase microextraction (in-tube SPME) technique to a tandem mass spectrometry system (MS/MS) to determine amino acids (AA) and neurotransmitters (NT) (alanine, serine, isoleucine, leucine, aspartic acid, glutamic acid, lysine, methionine, tyrosine, and tryptophan) in plasma samples from schizophrenic patients. An innovative organic-silica hybrid monolithic capillary with bifunctional groups (amino and cyano) was developed and evaluated as an extraction device for in-tube SPME. The morphological and structural aspects of the monolithic phase were evaluated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), nitrogen sorption experiments, X-ray diffraction (XRD) analyses, and adsorption experiments. In-tube SPME-MS/MS conditions were established to remove matrix, enrich analytes (monolithic capillary) and improve the sensitivity of the MS/MS system. The proposed method was linear from 45 to 360 ng mL -1 for alanine, from 15 to 300 ng mL -1 for leucine and isoleucine, from 12 to 102 ng mL -1 for methionine, from 10 to 102 ng mL -1 for tyrosine, from 9 to 96 ng mL -1 for tryptophan, from 12 to 210 ng mL -1 for serine, from 12 to 90 ng mL -1 for glutamic acid, from 12 to 102 ng mL -1 for lysine, and from 6 to 36 ng mL -1 for aspartic acid. The precision of intra-assays and inter-assays presented CV values ranged from 1.6% to 14.0%. The accuracy of intra-assays and inter-assays presented RSE values from -11.0% to 13.8%, with the exception of the lower limit of quantification (LLOQ) values. The in-tube SPME-MS/MS method was successfully applied to determine the target AA and NT in plasma samples from schizophrenic patients.

Ancient protein analysis is a rapidly developing field of research. Proteins ranging in age from the Quaternary to Jurassic are being used to answer questions about phylogeny, evolution, and extinction. However, these analyses are sometimes contentious, and focus primarily on large vertebrates in sedimentary fossilisation environments; there are few studies of protein preservation in fossils in amber. Here we show exceptionally slow racemisation rates during thermal degradation experiments of resin enclosed feathers, relative to previous thermal degradation experiments of ostrich eggshell, coral skeleton, and limpet shell. We also recover amino acids from two specimens of fossil feathers in amber. The amino acid compositions are broadly similar to those of degraded feathers, but concentrations are very low, suggesting that much of the original protein has been degraded and lost. High levels of racemisation in more apolar, slowly racemising amino acids suggest that some of the amino acids were ancient and therefore original. Our findings indicate that the unique fossilisation environment inside amber shows potential for the recovery of ancient amino acids and proteins.

Trichosanthes kirilowii Maxim. is one of the original plants for traditional Chinese medicines Trichosanthis Fructus, Trichosanthis Semen, Trichosanthis Pericarpium and Trichosanthis Radix. Amino acids, nucleosides and carbohydrates are usually considered to have nutritional value and health-care efficacy. In this study, methods involving high-performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD), UV-visible spectrophotometry and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) were established for quantifying carbohydrates (fructose, glucose, stachyose, raffinose and polysaccharide), fourteen nucleosides and twenty one amino acids. Moreover, sixty-three samples from nine different parts, including pericarp, seed, fruit pulp, stem, leaf, main root, main root bark, lateral root and lateral root bark of T. kirilowii from different cultivated varieties were examined. The established methods were validated with good linearity, precision, repeatability, stability, and recovery. The results showed that the average content of total amino acids in roots (15.39 mg/g) and root barks (16.38 mg/g) were relatively higher than for others. Contents of nucleosides in all parts of T. kirilowii were below 1.5 mg/g. For carbohydrates, fruit pulp has a higher content than others for glucose (22.91%), fructose (20.63%) and polysaccharides (27.29%). By using partial least-squared discriminate analysis (PLS-DA), Variable importance in the projection (VIP) plots and analysis of variance (ANOVA) analysis, the characteristic components of the different organs (fruit, stems and leaves, roots) were found. This analysis suggested there were potential medicinal and nutritive health care values in various parts of the T. kirilowii , which provided valuable information for the development and utilization of T. kirilowii .

The use of nanomaterials in bioseparations has been recently introduced to overcome the drawbacks of the conventional methods. Different forms of nanomaterials, particularly magnetic nanoparticles (MNPs), carbon nanotubes (CNTs), casted nanoporous membranes, and electrospun nanofiber membranes were utilized in biological separation for the aim of production of different biomolecules such as proteins, amino acids, nucleic acids, and enzymes. This paper critically reviews the state-of-the-art efforts undertaken in this regard, with emphasis on the synthesis and performance evaluation of each nanoform. Challenges and future prospects in developing nanoenabled bioseparations are also discussed, for the purpose of highlighting potential advances in the synthesis and fabrication of novel nanomaterials as well as in the design of efficient nanoenabled processes for separating a wide spectrum of biomolecules.

The safety and stability of synthetic UV-filters and the procedures for evaluating the photoprotective capability of commercial sunscreens are under continuous review. The influence of pH and temperature stressors on the stability of certain Mycosporine-like amino acids (MAAs) isolated at high purity levels was examined. MAAs were highly stable at room temperature during 24 h at pH 4.5⁻8.5. At 50 °C, MAAs showed instability at pH 10.5 while at 85 °C, progressive disappearances were observed for MAAs through the studied pH range. In alkaline conditions, their degradation was much faster. Mycosporine-serinol and porphyra-334 (+shinorine) were the most stable MAAs under the conditions tested. They were included in four cosmetically stable topical sunscreens, of which the Sun Protection Factor (SPF) and other Biological Effective Protection Factors (BEPFs) were calculated. The formulation containing these MAAs showed similar SPF and UVB-BEPFs values as those of the reference sunscreen, composed of synthetic UV absorbing filters in similar percentages, while UVA-BEPFs values were slightly lower. Current in vitro data strongly suggest that MAAs, as natural and safe UV-absorbing and antioxidant compounds, have high potential for protection against the diverse harmful effects of solar UV radiation. In addition, novel complementary in vitro tests for evaluation of commercial sunscreens efficacy are proposed.

Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.

Marine algae have received great attention as natural photoprotective agents due to their unique and exclusive bioactive substances which have been acquired as an adaptation to the extreme marine environment combine with a range of physical parameters. These photoprotective substances include mycosporine-like amino acids (MAAs), sulfated polysaccharides, carotenoids, and polyphenols. Marine algal photoprotective substances exhibit a wide range of biological activities such as ultraviolet (UV) absorbing, antioxidant, matrix-metalloproteinase inhibitors, anti-aging, and immunomodulatory activities. Hence, such unique bioactive substances derived from marine algae have been regarded as having potential for use in skin care, cosmetics, and pharmaceutical products. In this context, this contribution aims at revealing bioactive substances found in marine algae, outlines their photoprotective potential, and provides an overview of developments of blue biotechnology to obtain photoprotective substances and their prospective applications.

Non-volatile organic acids and amino acids are important flavor compounds in Pixian broad-bean paste, which is a traditional Chinese seasoning product. In this study, non-volatile organic acids, formed in the broad-bean paste due to the metabolism of large molecular compounds, are qualitatively and quantitatively determined by high-performance liquid chromatography (HPLC). Amino acids, mainly produced by hydrolysis of soybean proteins, were determined by the amino acid automatic analyzer. Results indicated that seven common organic acids and eighteen common amino acids were found in six Pixian broad-bean paste samples. The content of citric acid was found to be the highest in each sample, between 4.1 mg/g to 6.3 mg/g, and malic acid were between 2.1 mg/g to 3.6 mg/g ranked as the second. Moreover, fumaric acid was first detected in fermented bean pastes albeit with a low content. For amino acids, savory with lower sour taste including glutamine (Gln), glutamic acid (Glu), aspartic acid (Asp) and asparagines (Asn) were the most abundant, noted to be 6.5 mg/g, 4.0 mg/g, 6.4 mg/g, 4.9 mg/g, 6.2 mg/g and 10.2 mg/g, and bitter taste amino acids followed. More importantly, as important flavor materials in Pixian broad-bean paste, these two groups of substances are expected to be used to evaluate and represent the flavor quality of Pixian broad-bean paste. Moreover, the results revealed that citric acid, glutamic acid, methionine and proline were the most important flavor compounds. These findings are agreat contribution for evaluating the quality and further assessment of Pixian broad-bean paste.

The combination of LC-MS/MS based metabolomics approach and anti-MRSA activity-guided fractionation scheme was applied on the Gram-negative bacterium Aequorivita sp. isolated from shallow Antarctic sea sediment using a miniaturized culture chip technique. This methodology afforded the isolation of three new ( 1 ⁻ 3 ) and four known ( 4 ⁻ 7 ) N-terminal glycine- or serine-bearing iso -fatty acid amides esterified with another iso -fatty acid through their C-3 hydroxy groups. The chemical structures of the new compounds were elucidated using a set of spectroscopic (NMR, [α] D and FT-IR) and spectrometric (HRMS, HRMS/MS) methods. The aminolipids possessing an N-terminal glycine unit ( 1 , 2 , 4 , 5 ) showed moderate in vitro antimicrobial activity against MRSA (IC 50 values 22⁻145 μg/mL). This is the first in-depth chemistry and biological activity study performed on the microbial genus Aequorivita .

A solid biostimulant (AA309) obtained through thermobaric hydrolysis applied on trimmings and shavings of bovine hides tanned with wet-blue technology was chemically characterized, and its effects in maize ( Zea mays L.) were evaluated. AA309 contained 13.60% total nitrogen (N), mainly in organic forms (13.40%), and several amino acids, especially lysine, phenylalanine, glycine, aspartate, and isoleucine. AA309 was further analyzed using Fourier Transform Infrared (FT-IR) spectroscopy, which revealed the presence of amide I and amide II bands, indicative of peptide structures. When supplied to maize plants for 15 days at two N dosages (2.1 or 4.2 mg/kg), AA309 induced positive physiological responses, likely because of its content in amino acids functioning as signaling molecules. The low dosage was the most effective in improving leaf (+24%) and root (+98%) dry weight, photosynthetic activity (+70%), and accumulation of N (+80%), proteins (+65⁻75%) and antioxidants (+60%). Spectroscopic analyses (Solid-state Cross-Polarization Magic Angle Spinning Carbon-13 Nuclear Magnetic Resonance, CP/MAS 13 C⁻NMR, and High resolution-magic angle spinning nuclear magnetic resonance, HR-MAS NMR) on plant tissues revealed the increase in proteins, lignin structures and cutin in AA309-treated plants compared to untreated plants. Our results indicate that AA309 could be used as a valuable biostimulant in agriculture.

Apocyni Veneti Folium (AVF) is a kind of staple traditional Chinese medicine with vast clinical consumption because of its positive effects. However, due to the habitats and adulterants, its quality is uneven. To control the quality of this medicinal herb, in this study, the quality of AVF was evaluated based on simultaneous determination of multiple bioactive constituents combined with multivariate statistical analysis. A reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was developed for the simultaneous determination of a total of 43 constituents, including 15 flavonoids, 6 organic acids, 13 amino acids, and 9 nucleosides in 41 Luobumaye samples from different habitats and commercial herbs. Furthermore, according to the contents of these 43 constituents, principal component analysis (PCA) was employed to classify and distinguish between AVF and its adulterants, leaves of Poacynum hendersonii (PHF), and gray relational analysis (GRA) was performed to evaluate the quality of the samples. The proposed method was successfully applied to the comprehensive quality evaluation of AVF, and all results demonstrated that the quality of AVF was higher than the PHF. This study will provide comprehensive information necessary for the quality control of AVF., Competing Interests: The authors declare no conflicts of interest.

The primary metabolites in aqueous extract of mulberry ( Morus alba L.) leaves were characterized by using proton nuclear magnetic resonance (¹H-NMR) spectroscopy. With the convenience of resonance assignment, GABA together with the other 10 primary metabolites was simultaneously identified and quantified in one ¹H-NMR spectrum. In this study, external calibration curves for metabolites were employed to calculate the concentrations of interests. The proposed quantitative approach was demonstrated with good linearity ( r² ranged in the interval of 0.9965-0.9999), precision, repeatability, stability (RSD values in the ranges of 0.35-4.89%, 0.77-7.13% and 0.28-2.33%, respectively) and accuracy (recovery rates from 89.2% to 118.5%). The established ¹H-NMR method was then successfully applied to quantify 11 primary metabolites in mulberry leaves from different geographical regions within a rapid analysis time and a simple sample preparation procedure., Competing Interests: There are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Inflammation and oxidative stress play major roles in endothelial dysfunction, and are key factors in the progression of cardiovascular diseases. The aim of this study was to evaluate in vitro the effect of three subfractions (SFs) from the Cucumis sativus aqueous fraction to reduce inflammatory factors and oxidative stress induced by angiotensin II (Ang II) in human microvascular endothelial cells-1 (HMEC-1) cells. The cells were cultured with different concentrations of Ang II and 0.08 or 10 μg/mL of SF1, SF2, or SF3, or 10 μmol of losartan as a control. IL-6 (Interleukin 6) concentration was quantified. To identify the most effective SF combinations, HMEC-1 cells were cultured as described above in the presence of four combinations of SF1 and SF3. Then, the effects of the most effective combination on the expression of adhesion molecules, the production of reactive oxygen species (ROS), and the bioavailability of nitric oxide (NO) were evaluated. Finally, a mass spectrometry analysis was performed. Both SF1 and SF3 subfractions decreased the induction of IL-6 by Ang II, and C4 (SF1 and SF3, 10 μg/mL each) was the most effective combination to inhibit the production of IL-6. Additionally, C4 prevented the expression of adhesion molecules, reduced the production of ROS, and increased the bioavailability of NO. Glycine, arginine, asparagine, lysine, and aspartic acid were the main components of both subfractions. These results demonstrate that C4 has anti-inflammatory and antioxidant effects., Competing Interests: The authors declare no conflict of interest.

Huanglongbing is a devastating disease of citrus. In this study, a comprehensive profile of phloem sap amino acids (AA) in four permissive host plants of Candidatus Liberibacter asiaticus (CLas) and three non-permissive Rutaceae plants was conducted to gain a better understanding of host factors that may promote or suppress the bacterium. The AA profiles of Diaphorina citri nymphs and adults were similarly analyzed. A total of 38 unique AAs were detected in phloem sap of the various plants and D. citri samples, with phloem sap of young shoots containing more AAs and at higher concentrations than their mature counterparts. All AAs detected in phloem sap of non-permissive plants were also present in CLas -permissive hosts plus additional AAs in the latter class of plants. However, the relative composition of 18 commonly shared AAs varied between CLas -permissive hosts and non-permissive plants. Multivariate analysis with a partial least square discriminant methodology revealed a total of 12 AAs as major factors affecting CLas host status, of which seven were positively related to CLas tolerance/resistance and five positively associated with CLas susceptibility. Most of the AAs positively associated with CLas susceptibility were predominantly of the glutamate family, notably stressed-induced AAs such as arginine, GABA and proline. In contrast, AAs positively correlated with CLas tolerance/resistance were mainly of the serine family. Further analysis revealed that whereas the relative proportions of AAs positively associated with CLas susceptibility did not vary with host developmental stages, those associated with CLas tolerance/resistance increased with flush shoot maturity. Significantly, the proline-to-glycine ratio was determined to be an important discriminating factor for CLas permissivity with higher values characteristic of CLas -permissive hosts. This ratio could be exploited as a biomarker in HLB-resistance breeding programs.

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a group of rare progressive neurodegenerative disorders caused by an abnormally folded prion protein (PrP Sc ). This is capable of transforming the normal cellular prion protein (PrP C ) into new infectious PrP Sc Interspecies prion transmissibility studies performed by experimental challenge and the outbreak of bovine spongiform encephalopathy that occurred in the late 1980s and 1990s showed that while some species (sheep, mice, and cats) are readily susceptible to TSEs, others are apparently resistant (rabbits, dogs, and horses) to the same agent. To study the mechanisms of low susceptibility to TSEs of certain species, the mouse-rabbit transmission barrier was used as a model. To identify which specific amino acid residues determine high or low susceptibility to PrP Sc propagation, protein misfolding cyclic amplification (PMCA), which mimics PrP C -to-PrP Sc conversion with accelerated kinetics, was used. This allowed amino acid substitutions in rabbit PrP and accurate analysis of misfolding propensities. Wild-type rabbit recombinant PrP could not be misfolded into a protease-resistant self-propagating isoform in vitro despite seeding with at least 12 different infectious prions from diverse origins. Therefore, rabbit recombinant PrP mutants were designed to contain every single amino acid substitution that distinguishes rabbit recombinant PrP from mouse recombinant PrP. Key amino acid residue substitutions were identified that make rabbit recombinant PrP susceptible to misfolding, and using these, protease-resistant misfolded recombinant rabbit PrP was generated. Additional studies characterized the mechanisms by which these critical amino acid residue substitutions increased the misfolding susceptibility of rabbit PrP. IMPORTANCE Prion disorders are invariably fatal, untreatable diseases typically associated with long incubation periods and characteristic spongiform changes associated with neuronal loss in the brain. Development of any treatment or preventative measure is dependent upon a detailed understanding of the pathogenesis of these diseases, and understanding the mechanism by which certain species appear to be resistant to TSEs is critical. Rabbits are highly resistant to naturally acquired TSEs, and even under experimental conditions, induction of clinical disease is not easy. Using recombinant rabbit PrP as a model, this study describes critical molecular determinants that confer this high resistance to transmissible spongiform encephalopathies., (Copyright © 2017 American Society for Microbiology.)

Background/aim: Most of the previous investigators have used various types of media for the culture of nerve cells. In order to optimize the culture conditions, we compared the growth rate and amino acid consumption by two popular neuron models, rat PC12 and human SH-SY5Y, grown in DMEM or DMEM: Ham's F-12 (1:1): non-essential amino acids, supplemented with 10% fetal bovine serum (referred to DMEM and Mix, respectively)., Materials and Methods: Cell growth was monitored by the MTT method. Amino acids in the culture medium were quantitated by amino acid analysis after deproteinization., Results: Efficient cell attachment could be achieved even if PC12 cells were inoculated at extreme lower cell density in a non-coated plain dish, without addition of its condition medium. Both PC12 and SH-SY5Y cells proliferated up to slightly higher cell density in DMEM than in Mix. Approximately 2-fold higher utilization rate of glutamine and essential amino acids was observed in DMEM. Amyloid peptides such as Aβ 1-42 and Aβ 25-35 suppressed their growth nearly by 50%., Conclusion: The present study suggests the usefulness of DMEM for the study of searching neuroprotective substances, based on its favorable effects on cell attachment, cell growth and amino acid utilization as well as amyloid peptide sensitivity., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

In this study, we optimized a method for the determination of free amino acids in Nicotiana tabacum leaves. Capillary electrophoresis with contactless conductivity detector was used for the separation of 20 proteinogenic amino acids in acidic background electrolyte. Subsequently, the conditions of extraction with HCl were optimized for the highest extraction yield of the amino acids because sample treatment of plant materials brings some specific challenges. Central composite face-centered design with fractional factorial design was used in order to evaluate the significance of selected factors (HCl volume, HCl concentration, sonication, shaking) on the extraction process. In addition, the composite design helped us to find the optimal values for each factor using the response surface method. The limits of detection and limits of quantification for the 20 proteinogenic amino acids were found to be in the order of 10 -5 and 10 -4  mol l -1 , respectively. Addition of acetonitrile to the sample was tested as a method commonly used to decrease limits of detection. Ambiguous results of this experiment pointed out some features of plant extract samples, which often required specific approaches. Suitability of the method for metabolomic studies was tested by analysis of a real sample, in which all amino acids, except for L-methionine and L-cysteine, were successfully detected. The optimized extraction process together with the capillary electrophoresis method can be used for the determination of proteinogenic amino acids in plant materials. The resulting inexpensive, simple, and robust method is well suited for various metabolomic studies in plants. As such, the method represents a valuable tool for research and practical application in the fields of biology, biochemistry, and agriculture.

Pseudostellariae Radix (PR) is an important traditional Chinese herbal medicine (TCM) with vast clinical consumption because of its positive effects. However, little attention has been devoted to simultaneous analysis of its bioactive components for quality control of PR based on its different harvesting times, different growing habitats, and different processing methods. In this research, the quality of PR was evaluated based on simultaneous determination of multiple bioactive components combined with grey relational analysis (GRA). A reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established to simultaneously determine the contents of 30 components in PR, including two cyclopeptides, 12 nucleosides, and 16 amino acids. Furthermore, grey relational analysis was performed to evaluate the quality of PR samples according to the contents of these 30 components. The results showed that the quality of PR harvested in 6 August 2013, cultivated in Jurong, Jiangsu, and treated by oven drying 60 °C was better than that of other PR samples. The proposed method is useful for the overall assessment on the quality of PR, and this study provides valuable information for revealing the dynamic change laws of metabolite accumulation in PR and choosing the most suitable harvesting time and reasonable processing method of PR to obtain the best quality.

"Inherently chiral" thiophene-based electroactive oligomer films have recently been shown to exhibit outstanding chirality manifestations. One of the most exciting among them is an unprecedented enantioselection ability as electrode surfaces. In fact, in preliminary chiral voltammetry experiments, the new electrodes have been shown to both discriminate the enantiomers of chiral probes (either enantiopure or in a mixture, in terms of large differences in peak potentials) and quantify them (in terms of linear dynamic ranges in peak currents), without the need for preliminary separation steps. Such ability has now been tested on a series of chiral DOPA-related molecules, from phenolic amino acid tyrosine (together with its methyl ester) to catecholic amino acid DOPA (together with its methyl ester), to catecholamine epinephrine (adrenaline). The wide-range enantioselectivity of the new inherently chiral electrode surfaces is fully confirmed, as large peak potential differences are obtained for probe enantiomers of the whole series working in common aqueous buffers. Moreover, interesting modulating effects on enantiodiscrimination can be observed as a function of both molecular structure and pH. Graphical abstract Inherently chiral thiophene-based electrodes at work with pharmaceutically relevant probes.

In the current study, the functional and biochemical potential of the seeds of four persimmon cultivars (PC1, PC2, PC3 and PC4) and their role against oxidative stress and acetylcholinesterase (AChE) inhibition were evaluated. In terms of biochemical compositions, free amino acids, fatty acids and organic acids analysis was performed. The free amino acids ranged from 2617.31 (PC2) to 3773.01 μg∙g(-1) dry weight (PC4). Oleic acid and linoleic acid were the principal fatty acids, which were significantly higher in PC4 and PC1, respectively. PC4 presented the highest amount of organic acid content (4212 mg∙kg(-1)), whereas PC2 presented the lowest (2498 mg∙kg(-1)). PC2 contained higher total phenolic content and flavonoid content, whereas PC3 had the lowest amount as compared to other cultivars. The in vitro DPPH, ABTS and superoxide anion radicals scavenging activity increased in a dose-dependent manner, whereas PC2 showed significantly higher scavenging activities as compared to PC1, PC2 and PC4 types. In the case of AChE inhibition, PC4 showed a moderate activity (67.34% ± 1.8%). In conclusion, the current findings reveal that the studied persimmon seeds cultivars are a source of bioactive natural antioxidants and AChE inhibitors. Such natural products could be employed in pharmaceutical and food industries, whilst can also be considered for the treatment of neurodegenerative diseases such as Alzheimer's.

To evaluate the nutritional and functional value of Sipunculus nudus, a rapid, simple and sensitive analytical method was developed using ultra-high performance liquid chromatography coupled with a triple quadrupole mass detection in multiple-reaction monitoring mode for the simultaneous quantitative determination of 25 free amino acids and 16 nucleosides and nucleobases in S. nudus within 20 min, which was confirmed to be reproducible and accurate. The limits of detection (LODs) and quantification (LOQs) were between 0.003-0.229 μg/mL and 0.008-0.763 μg/mL for the 41 analytes, respectively. The established method was applied to analyze 19 batches of S. nudus samples from four habitats with two different processing methods. The results showed that S. nudus contained a variety of free amino acids, nucleosides and nucleobases in sufficient quantity and reasonable proportion. They also demonstrated that the contents of these compounds in different parts of S. nudus were significantly discriminating, which were in the order: (highest) coelomic fluid > body wall > intestine (lowest). The method is simple and accurate, and could serve as a technical support for establishing quality control of S. nudus and other functional seafoods. Moreover, the research results also laid foundation for further exploitation and development of S. nudus.

An extra-facile chiral liquid chromatography-time of flight mass spectrometry (LC-TOFMS) analytical method of amino acid enantiomers has been developed without a derivatization process. The enantioseparation of eighteen proteinogenic amino acids (except for proline) was simultaneously performed using a combination of a chiral column (CROWNPAK CR-I(+)) and a TOFMS within 15 min. An isocratic condition of a simple mobile phase comprising acetonitrile/water/trifluoroacetic acid (96/4/0.5) gave baseline separation of all underivatized amino acid enantiomers on the chiral column., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Enantioseparation of D, L-amino acids is of great significance in life science. As one of the most useful methods, chiral ligand exchange capillary electrophoresis possesses many advantages, including high efficiency, fast speed and tunable migration order, and it has attracted great research interest. This review summarizes the development of chiral ligand exchange capillary electrophoresis for enantioseparation of D, L-amino acids in recent years.

A novel chiral stationary phase combining quinine and crown ether (QN-CR CSP) was developed to separate amino acid enantiomers. This CSP showed good enantioselectivity for some amino acids. Since the synergistic effect of ion exchange and complexation in chiral recognition of amino acids, a new adsorption isotherm was built. Using the method of frontal analysis by characteristic point (FACP), the adsorption isotherms of tryptophan (Trp) under different mobile phase conditions were determined and fitted the proposed adsorption isotherm model well. With the increase of the competition between metal cationic and amino to crown ether, the equilibrium constant of complexing adsorption was found increased. The chiral separation ability was decreased. The adsorption isotherm improved the understanding of the retention behavior of amino acids on QN-CR CSP, which was also benefit to optimize the structure of the stationary phase.

The objective of this study was to elucidate the mixing state of proteins and amino acid excipients concentrated in the amorphous non-ice region of frozen solutions. Thermal analysis of frozen aqueous solutions was performed in heating scans before and after a heat treatment. Frozen aqueous solutions containing a protein (e.g., recombinant human albumin, gelatin) or a polysaccharide (dextran) and an amino acid excipient (e.g., L-arginine, L-arginine hydrochloride, L-arginine monophosphate, sodium L-glutamate) at varied mass ratios showed single or double T g ' (glass transition temperature of maximally freeze-concentrated solutes). Some mixture frozen solutions rich in the polymers maintained the single T g ' of the freeze-concentrated amorphous solute-mixture phase. In contrast, amino acid-rich mixture frozen solutions revealed two T g 's that suggested transition of concentrated non-crystalline solute-mixture phase and excipient-dominant phase. Post-freeze heat treatment induced splitting of the T g ' in some intermediate mass ratio mixture solutions. The mixing state of proteins and amino acids varied depending on their structure, salt types, mass ratio, composition of co-solutes (e.g., NaCl) and thermal history. Information on the varied mixing states should be valuable for the rational use of amino acid excipients in lyophilized protein pharmaceuticals.

In this study, five common buckwheats and nine tartary buckwheats grown at different locations were analyzed for the contents of rutin, quercetin, and amino acids by high-performance liquid chromatography and spectrophotometry. The rutin content was higher than quercetin in buckwheat seeds. Rutin content was in the range from 0.05 (0.05 g per 100 g dry seeds) to 1.35% of buckwheat seeds. Quercetin content varied from 0.01 to 0.17% and in some common buckwheats it was even difficult to detect. Comparatively, tartary buckwheat seeds contained more rutin and quercetin than common buckwheat seeds. Meanwhile, the bran has higher rutin content than the farina in tartary buckwheat seeds, with a respective content of 0.45 to 1.19% and 0.14 to 0.67%. It was found that amino acid contents were around 1.79 to 12.65% (farina) and 5.74 to 7.89% (bran) in common buckwheats, and 1.73 to 5.63% (farina) and 2.64 to 16.78% (bran) in tartary buckwheat seeds. The highest total rutin content was found to be 1.35% in tartary buckwheat seeds from Sichuan, China. The highest total amounts of amino acid were detected to be 20.13% in tartary buckwheat seeds from Changzhi, Shanxi Province (China). Our results suggested that food products made of whole-buckwheat flour are healthier than those made of fine white flour.

There is a strong drive worldwide to discover and exploit the therapeutic potential of a large variety of plants. In this work, an alcoholic extract of Helleborus purpurascens (family Ranunculaceae) was investigated for the identification of amino acids and peptides with putative antiproliferative effects. In our work, a separation strategy was developed using solvents of different polarity in order to obtain active compounds. Biochemical components were characterized through spectroscopic (mass spectroscopy) and chromatographic techniques (RP-HPLC and GC-MS). The biological activity of the obtained fractions was investigated in terms of their antiproliferative effects on HeLa cells. Through this study, we report an efficient separation of bioactive compounds (amino acids and peptides) from a plant extract dependent on solvent polarity, affording fractions with unaffected antiproliferative activities. Moreover, the two biologically tested fractions exerted a major antiproliferative effect, thereby suggesting potential anticancer therapeutic activity.

Mycosporine-like amino acids (MAAs), a group of small secondary metabolites found in algae, cyanobacteria, lichens and fungi, have become ecologically and pharmacologically relevant because of their pronounced UV-absorbing and photo-protective potential. Their analytical characterization is generally achieved by reversed phase HPLC and the compounds are often quantified based on molar extinction coefficients. As an alternative approach, in our study a fully validated hydrophilic interaction liquid chromatography (HILIC) method is presented. It enables the precise quantification of several analytes with adequate retention times in a single run, and can be coupled directly to MS. Excellent linear correlation coefficients (R² > 0.9991) were obtained, with limit of detection (LOD) values ranging from 0.16 to 0.43 µg/mL. Furthermore, the assay was found to be accurate (recovery rates from 89.8% to 104.1%) and precise (intra-day precision: 5.6%, inter-day precision ≤6.6%). Several algae were assayed for their content of known MAAs like porphyra-334, shinorine, and palythine. Liquid chromatography-mass spectrometry (LC-MS) data indicated a novel compound in some of them, which could be isolated from the marine species Catenella repens and structurally elucidated by nuclear magnetic resonance spectroscopy (NMR) as (E)-3-hydroxy-2-((5-hydroxy-5-(hydroxymethyl)-2-methoxy-3-((2-sulfoethyl)amino)cyclohex-2-en-1-ylidene)amino) propanoic acid, a novel MAA called catenelline.

In this work, a straightforward in-situ measurement of L and D-amino acids (AAs) has been developed using disposable graphene oxide nanoribbon (GON) screen printed electrodes. For that, we took advantage of the electroactivity of certain clinically relevant AAs, such as tyrosine (Tyr) and methionine (Met), which are involved in important bacterial diseases (Bacillus subtilis and Vibrio cholera, respectively). The strategy is based on a dual electrochemical and enzymatic approach. The D-AA with the class enzyme D amino acid oxidase (DAAO) generates H2O2. This H2O2 is simultaneously detected with the L-AA, electroactive molecule by differential pulse voltammetry (DPV). These GON disposable platforms use just 50 μL of sample and a total analysis time of 360 s. Both L and D enantiomers calibration and quantitative analysis were explored and were simultaneously detected with accuracy and precision in urine samples. Any interference of uric acid and other electroactive AAs was noticed. This proposed electrochemical GON-based enantiomeric bio-sensor becomes a highly promising tool as future point of care for fast and reliable early diagnosis of diseases related to the presence of D-AAs., (Copyright © 2014 Elsevier B.V. All rights reserved.)