UHPLC-PDA-Q-TOF-MS-α-amylase-FLD activity detection system and molecular docking.

INTRODUCTION: Traditional and some scientific literature document the antidiabetic effects of the Ziziphi Spinosae Semen (ZSS). However, the bioactive compounds of ZSS responsible for the antidiabetic effects are not well known. OBJECTIVES: This study aimed to investigate the material basis of the antidiabetic effects of ZSS by inhibiting α-amylase. METHODOLOGY: An online analysis platform was established and optimized using an ultra-performance liquid chromatography-photo-diode array-quadrupole-time-of-flight-mass spectrometry-α-amylase-fluorescence detector (UHPLC-PDA-Q-TOF-MS-α-amylase-FLD) system to screen α-amylase inhibitors in ZSS rapidly. The inhibitory effect of these compounds was confirmed by molecular docking screening. and the molecular interactions between α-amylase and active compounds were evaluated, which strongly supported the experimental results. RESULTS: Seventy-eight compounds were identified in the ZSS extract, eleven of which were screened to have significant α-amylase binding activity. CONCLUSION: This study demonstrated the feasibility of using an established platform to screen for effective components in ZSS, providing a practical method for the rapid screening of potential antidiabetic active ingredients in traditional Chinese medicine.

Strategy for the quality control of herbal preparations made of Sarcocephalus latifolius: Development and validation of a UHPLC-PDA method for quantification of angustoline and strictosamide and chemical profiling using LC-QToF.

INTRODUCTION: Sarcocephalus latifolius is one of the most used plants in West African traditional medicine to treat malaria. OBJECTIVE: The aim is to establish a strategy to control the quality of herbal preparations made from S. latifolius. METHOD: A UHPLC-PDA method was developed for the determination and quantification of the two main bioactive compounds (angustoline and strictosamide) in various parts of the plant. Additionally, an LC-QToF with electrospray ionization method is described for the identification and confirmation of compounds in samples of different parts of the plant. RESULTS: With the UHPLC-PDA method, separation was achieved within 5 min using a C18 column stationary phase at a temperature of 45°C and a gradient system with a mobile phase of water and acetonitrile, both containing 0.1% formic acid. The method was validated for linearity, accuracy, precision (repeatability and intermediate precision), limit of detection (LOD), and limit of quantification (LOQ). The LOD and LOQ of angustoline were found to be 0.3 and 0.8 µg/ml, respectively, and those of strictosamide were found to be 0.1 and 0.3 µg/ml, respectively. Using the LC-QToF method, 90 secondary metabolites, including four isolated compounds from the plant's roots, were identified from leaf, bark, and root samples of S. latifolius. CONCLUSION: This work is the first to propose a strategy to control the quality of herbal preparations made from S. latifolius. The developed method allows the quantification of the main bioactive compounds and the established chemical profile allows to distinguish the plant from any other species.

Bioactive Compounds and Antioxidant Activity from Spent Coffee Grounds as a Powerful Approach for Its Valorization.

Coffee is one of the world's most popular beverages, and its consumption generates copious amounts of waste. The most relevant by-product of the coffee industry is the spent coffee grounds, with 6 million tons being produced worldwide per year. Although generally treated as waste, spent coffee grounds are a rich source of several bioactive compounds with applications in diverse industrial fields. The present work aimed at the analysis of spent coffee grounds from different geographical origins (Guatemala, Colombia, Brazil, Timor, and Ethiopia) for the identification of bioactive compounds with industrial interest. For this purpose, the identification and quantification of the bioactive compounds responsible for the antioxidant activity attributed to the spent coffee grounds were attempted using miniaturized solid-phase extraction (µ-SPEed), combined with ultrahigh-performance liquid chromatography with photodiode array detection (UHPLC-PDA). After validation of the µ-SPEed/UHPLC-PDA method, this allowed us to conclude that caffeine and 5-caffeoylquinic acid (5-CQA) are the most abundant bioactive compounds in all samples studied. The total phenolic content (TPC) and antioxidant activity are highest in Brazilian samples. The results obtained show that spent coffee grounds are a rich source of bioactive compounds, supporting its bioprospection based on the circular economy concept closing the loop of the coffee value chain, toward the valorization of coffee by-products.

Chemical Fingerprinting and Biological Evaluation of the Endemic Chilean Fruit Greigia sphacelata (Ruiz and Pav.) Regel (Bromeliaceae) by UHPLC-PDA-Orbitrap-Mass Spectrometry.

Greigia sphacelata (Ruiz and Pav.) Regel (Bromeliaceae) is a Chilean endemic plant popularly known as "quiscal" and produces an edible fruit consumed by the local Mapuche communities named as "chupón". In this study, several metabolites including phenolic acids, organic acids, sugar derivatives, catechins, proanthocyanidins, fatty acids, iridoids, coumarins, benzophenone, flavonoids, and terpenes were identified in G. sphacelata fruits using ultrahigh performance liquid chromatography-photodiode array detection coupled with a Orbitrap mass spectrometry (UHPLC-PDA-Orbitrap-MS) analysis for the first time. The fruits showed moderate antioxidant capacities (i.e., 487.11 ± 26.22 µmol TE/g dry weight) in the stable radical DPPH assay, 169.08 ± 9.81 TE/g dry weight in the ferric reducing power assay, 190.32 ± 6.23 TE/g dry weight in the ABTS assay, and 76.46 ± 3.18% inhibition in the superoxide anion scavenging assay. The cholinesterase inhibitory potential was evaluated against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). From the findings, promising results were observed for pulp and seeds. Our findings suggest that G. sphacelata fruits are a rich source of diverse secondary metabolites with antioxidant capacities. In addition, the inhibitory effects against AChE and BChE suggest that natural products or food supplements derived from G. sphacelata fruits are of interest for their neuroprotective potential.

Rapid UHPLC-MS metabolite profiling and phenotypic assays reveal genotypic impacts of nitrogen supplementation in oats.

INTRODUCTION: Oats (Avena sativa L.) are a whole grain cereal recognised for their health benefits and which are cultivated largely in temperate regions providing both a source of food for humans and animals, as well as being used in cosmetics and as a potential treatment for a number of diseases. Oats are known as being a cereal source high in dietary fibre (e.g. ß-glucans), as well as being high in antioxidants, minerals and vitamins. Recently, oats have been gaining increased global attention due to their large number of beneficial health effects. Consumption of oats has been proven to lower blood LDL cholesterol levels and blood pressure, thus reducing the risk of heart disease, as well as reducing blood-sugar and insulin levels. OBJECTIVES: Oats are seen as a low input cereal. Current agricultural guidelines on nitrogen application are believed to be suboptimal and only consider the effect of nitrogen on grain yield. It is important to understand the role of both variety and of crop management in determining nutritional quality of oats. In this study the response of yield, grain quality and grain metabolites to increasing nitrogen application to levels greater than current guidelines were investigated. METHODS: Four winter oat varieties (Mascani, Tardis, Balado and Gerald) were grown in a replicated nitrogen response trial consisting of a no added nitrogen control and four added nitrogen treatments between 50 and 200 kg N ha-1 in a randomised split-plot design. Grain yield, milling quality traits, ß-glucan, total protein and oil content were assessed. The de-hulled oats (groats) were also subjected to a rapid Ultra High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) metabolomic screening approach. RESULTS: Application of nitrogen had a significant effect on grain yield but there was no significant difference between the response of the four varieties. Grain quality traits however displayed significant differences both between varieties and nitrogen application level. ß-glucan content significantly increased with nitrogen application. The UHPLC-MS approach has provided a rapid, sub 15 min per sample, metabolite profiling method that is repeatable and appropriate for the screening of large numbers of cereal samples. The method captured a wide range of compounds, inclusive of primary metabolites such as the amino acids, organic acids, vitamins and lipids, as well as a number of key secondary metabolites, including the avenanthramides, caffeic acid, and sinapic acid and its derivatives and was able to identify distinct metabolic phenotypes for the varieties studied. Amino acid metabolism was massively upregulated by nitrogen supplementation as were total protein levels, whilst the levels of organic acids were decreased, likely due to them acting as a carbon skeleton source. Several TCA cycle intermediates were also impacted, potentially indicating increased TCA cycle turn over, thus providing the plant with a source of energy and reductant power to aid elevated nitrogen assimilation. Elevated nitrogen availability was also directed towards the increased production of nitrogen containing phospholipids. A number of both positive and negative impacts on the metabolism of phenolic compounds that have influence upon the health beneficial value of oats and their products were also observed. CONCLUSIONS: Although the developed method has broad applicability as a rapid screening method or a rapid metabolite profiling method and in this study has provided valuable metabolic insights, it still must be considered that much greater confidence in metabolite identification, as well as quantitative precision, will be gained by the application of higher resolution chromatography methods, although at a large expense to sample throughput. Follow up studies will apply higher resolution GC (gas chromatography) and LC (reversed phase and HILIC) approaches, oats will be also analysed from across multiple growth locations and growth seasons, effectively providing a cross validation for the results obtained within this preliminary study. It will also be fascinating to perform more controlled experiments with sampling of green tissues, as well as oat grains, throughout the plants and grains development, to reveal greater insight of carbon and nitrogen metabolism balance, as well as resource partitioning into lipid and secondary metabolism.

Comparison of different analytical techniques for the analysis of carotenoids in tamarillo (Solanum betaceum Cav.).

An on-line method based on the coupling of supercritical fluid extraction and supercritical fluid chromatography with triple quadrupole mass spectrometry detection (SFE-SFC-QqQ/MS) for the native carotenoids characterization and apocarotenoids detection in yellow tamarillo was developed for the first time; this is the first work reporting the application of this methodology for the apocarotenoids analysis in any matrix. An off-line liquid extraction (LE) and analysis by LC-PDA-MS, using both a conventional C30 (3 µm I.D. particles) and a novel C30 column with sub-2 micron particles were also performed. 31 compounds were extracted and identified by the developed SFE-SFC-QqQ/MS methodology in less than 18 min, including free carotenoids, carotenoids monoesters, carotenoids diesters and apocarotenoids in a very fast, and efficient way; moreover among those 31 compounds, 3 antheraxanthin monoesters and 9 apocarotenoids were detected in yellow tamarillo for the first time, namely apo-8'-zeaxanthinal, apo-10'-zeaxanthinal, apo-12'-zeaxanthinal, apo-14'-zeaxanthinal, apo-15'-zeaxanthinal, apo-12'-carotenal, apo-14'-carotenal and the two apocarotenoids esters apo-10'-zeaxanthinal-C4:0 and apo-8'-zeaxanthinal-C12:0. Further, the novel sub-2 micron particles C30 column, showed a better performance compared to the conventional C30 one. Finally a quantitative comparison of two selected carotenoids was performed by using SFE-SFC-QqQ/MS, SFC-QqQ/MS, and LC-PDA, which showed overall comparable results.

Phenolic Profile of Artemisia alba Turra.

The aim of this study was to evaluate flower and leaf methanol extracts of Artemisia alba Turra for their total phenolic and flavonoid contents, antioxidant capacity and to investigate their phenolic composition. The flower extract was richer in total phenolics and flavonoids and possessed higher antioxidant activity through DPPH and ABTS assays. The UHPLC-PDA-MS analysis of the flower and leaf methanol extracts revealed similar phenolic profile and allowed identification of 31 phenolic compounds (flavonoids, coumarins, and phenolic acids) by comparison with the respective reference compounds or tentatively characterized by their chromatographic behavior, UV patterns, and MS fragmentations. The presence of hispidulin, jaceosidin, desmethoxycentaureidin, and dicaffeoyl esters of quinic acid in A. alba is reported herein for the first time. The distribution of flavonoids in A. alba from different origins was discussed from chemotaxonomic point of view.

Supercritical CO2 Extraction of Nannochloropsis sp.: A Lipidomic Study on the Influence of Pretreatment on Yield and Composition.

Algal lipids have gained wide interest in various applications ranging from biofuels to nutraceuticals. Given their complex nature composed of different lipid classes, a deep knowledge between extraction conditions and lipid characteristics is essential. In this paper, we investigated the influence of different pretreatments on lipid extraction with supercritical CO2 by a lipidomic approach. Pretreatment was found to double the total extraction yield, thereby reaching 23.1 wt.% comparable to the 26.9 wt.% obtained with chloroform/methanol. An increase in acylglycerides was concurrently observed, together with a nearly doubling of free fatty acids indicative of partial hydrolysis. Moreover, an alteration in the distribution of glyco- and phospholipids was noted, especially promoting digalactosyldiglycerides and phosphatidylcholine as compared to monogalactosyldiglycerides and phosphatidylglycerol. At optimized conditions, supercritical CO2 extraction provided a lipid extract richer in neutral lipids and poorer in phospholipids as compared to chloroform/methanol, though with a very similar fatty acid distribution within each lipid class.

Flavonoid Composition of Tarocco (Citrus sinensis L. Osbeck) Clone "Lempso" and Fast Antioxidant Activity Screening by DPPH-UHPLC-PDA-IT-TOF.

INTRODUCTION: Clonal selection and hybridisation are valid strategies to obtain fruits with enhanced sensorial and nutraceutical properties. Within Citrus sinensis varieties, Tarocco clone "Lempso" is a typical product of the Calabria region (Italy) characterised by its red pulp. This is the first report concerning its accurate profiling. OBJECTIVE: To characterise in detail the flavonoid composition of Lempso clone and to compare its antioxidant potential with other Citrus varieties by a fast screening method. METHODOLOGY: Extracts were subjected to solid phase extraction and the qualitative/quantitative profile was elucidated through ultra-high performance liquid chromatography (UHPLC) coupled to photodiode array (PDA) and ion trap time-of-flight (IT-TOF) mass spectrometry detection, and compared to both Cleopatra mandarin (Citrus reticulata) and blood orange (Citrus sinensis (L.) Osbeck) Sanguinello varieties. The antioxidant activity was assessed by pre-column 2,2'-diphenyl-1-picrylhydrazyl (DPPH) reaction coupled to UHPLC-PDA. RESULTS: Lempso is characterised by flavonoids (17) and anthocyanins (8). Flavanones content (Hesperidin: 57.19 ± 0.49, Vicenin-2: 4.59 ± 0.03, Narirutin: 5.78 ± 0.13 mg/100 mL) was considerably higher than Cleopatra and Sanguinello varieties. The developed DPPH-UHPLC-PDA method provides information regarding the single contributions to antioxidant activity, highlighting how Ferulic acid, Quercetin and Cyanidin derivatives possess considerable radical scavenging activity (> 50%). The total antioxidant activity was also evaluated and compared with positive controls, showing higher scavenging activity than Cleopatra and Sanguinello (IC50 : 333.76 ± 10.81 µg/mL vs. 452.62 ± 10.81 and 568.39 ± 26.98 µg/mL, respectively). CONCLUSION: These data evidence the nutraceutical potential of Lempso variety, which could be an ingredient for functional beverages. Copyright © 2017 John Wiley & Sons, Ltd.

Phenolic Profile and Antioxidant Potential of Leaves from Selected Cotoneaster Medik. Species.

The antioxidant efficiency of 70% aqueous methanolic extracts from the leaves of twelve selected Cotoneaster Medik. species was evaluated using four complementary in vitro tests based on SET- (single electron transfer) and HAT-type (hydrogen atom transfer) mechanisms (DPPH, FRAP, O2(•-) and H2O2 scavenging assays). The samples exhibited the dose-dependent responses in all assays with activity parameters of EC50 = 18.5-34.5 µg/mL for DPPH; 0.9-3.8 mmol Fe(2+)/g for FRAP; SC50 = 27.7-74.8 µg/mL for O2(•-); and SC50 = 29.0-91.3 µg/mL for H2O2. Significant linear correlations (|r| = 0.76-0.97, p < 0.01) between activity parameters and total contents of phenolics (5.2%-15.4% GAE) and proanthocyanidins (2.1%-15.0% CYE), with weak or no effects for chlorogenic acid isomers (0.69%-2.93%) and total flavonoids (0.28%-1.40%) suggested that among the listed polyphenols, proanthocyanidins are the most important determinants of the tested activity. UHPLC-PDA-ESI-QTOF-MS analyses led to detection of 34 polyphenols, of which 10 B-type procyanidins, 5 caffeoylquinic acids and 14 flavonoids were identified. After cluster analysis of the data matrix, the leaves of Cotoneaster zabelii, C. splendens, C. bullatus, C. divaricatus, C. hjelmqvistii and C. lucidus were selected as the most promising sources of natural antioxidants, exhibiting the highest phenolic levels and antioxidant capacities, and therefore the greatest potential for pharmaceutical applications.

Screening of the anthocyanin profile and in vitro pancreatic lipase inhibition by anthocyanin-containing extracts of fruits, vegetables, legumes and cereals.

BACKGROUND: The phytotherapic treatment of overweight and/or moderate obesity is growing widely, thus there is a great interest towards the phenolic compounds of fruits and vegetables which may inhibit pancreatic lipase enzyme. In this study, we report the chemical composition and in vitro pancreatic lipase inhibitory activity of 13 freeze-dried anthocyanin-containing extracts of different Mediterranean plants: fruits (blood orange, pomegranate, blackberry, mulberry and sumac), citrus by-products (blood orange peel), citrus vegetative tissues (young lemon shoots); vegetables (red cabbage and violet cauliflower), legume seeds (black bean), cereals (black rice), and cereal processing by-products (black rice hull). Total phenols and anthocyanins were determined. Individual anthocyanins were identified by UHPLC-PDA-ESI/MSn . RESULTS: Results revealed a wide variation in the distribution of anthocyanin compounds. Blood orange and pomegranate juice extracts had the highest total anthocyanin content and exhibited the strongest inhibition of pancreatic lipase in vitro. CONCLUSION: Inhibitory activity was positively correlated with anthocyanin content. In appropriate formulations, anthocyanin-containing extracts could find a use as anti-obesity agents. © 2016 Society of Chemical Industry.

Anthocyanins in different Citrus species: an UHPLC-PDA-ESI/MSn -assisted qualitative and quantitative investigation.

BACKGROUND: Anthocyanins are water-soluble pigments belonging to the flavonoid family. They are typically present in the flesh and peel in the blood orange cultivars. Although blood orange young shoots and flowers are not anthocyanin-colored, lemon, citron, rangpur lime, and Meyer lemon young shoots and flowers exhibit marked pigmentation as a result of anthocyanins, demonstrating that anthocyanin biosynthesis in the Citrus genus is both tissue- and genotype-dependent. The present study aimed to examine the qualitative and quantitative anthocyanin profile of fruit and other tissues from different Citrus species. RESULTS: The presence of anthocyanin-pigmented stigmas in the young flowers of a blood orange tree (cv. 'Moro') was characterized and reported for the first time. The dominant pigments in blood orange fruits were cyanidin 3-glucoside and cyanidin 3-(6''-malonyl-glucoside), whereas different patterns were observed in the young shoots, flowers and peel tissues of different Citrus species. CONCLUSION: The present study is the first to report differentially expressed anthocyanin pigmentation patterns in different organs from several species of the genus Citrus. The results obtained could also represent a starting point for further investigations that aim to better understand which regulatory genes are involved in anthocyanin biosynthesis in the fruits, shoots and floral tissues of different Citrus species. © 2016 Society of Chemical Industry.

Metabolomics and quantitative analysis to determine differences in the geographical origins and species of Chinese dragon's blood.

Objective: The aim of this study was to comprehensively analyze the differences in Chinese dragon's blood (CDB), specifically Dracaena cochinchinensis and Dracaena cambodiana, from different geographical origins. Methods: Metabolomic analysis of CDB was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A reliable ultrahigh-performance liquid chromatography method with a photodiode array detector (UHPLC-PDA) was developed and applied for the quantitative analysis of 12 phenolic compounds in 51 batches of samples. Results: A total of 1394 metabolites were detected, of which 467 were identified as differentially accumulated metabolites. Multivariate analysis revealed that both origin and species had an effect on the composition of CDB, with greater variation between species. 19 phenolic compounds were selected as quality markers to distinguish D. cochinchinensis (Hdsp) from D. cambodiana (Hdca), and oppositin and spinoflavanone a were identified as quality markers to discriminate D. cochinchinensis samples from Hainan (Hdsp) and Guangxi Provinces (Gdc). Quantitative analysis indicated that four phenolic compounds, including loureirin D, 4H-1-benzopyran-4-one,2,3-dihydro-3,5,7-trihydroxy-3-[(4-methoxyphenyl)methyl]-,(R)-, loureirin B, and pterostilbene, showed significant differences between Gdc and Hdsp. Additionally, five phenolic compounds, namely resveratrol, loureirin D, pinostilbene, 4H-1-benzopyran-4-one,2,3-dihydro-3,5,7-trihydroxy-3-[(4-methoxyphenyl)methyl]-, (R)-, and loureirin B, exhibited significant differences between Hdsp and Hdca. Conclusion: There are significant differences in the quality of CDB from different geographical origins and species, which lays the foundation for the in-depth development and utilization of different sources of CDB.

Development and Validation of SI/RS-UHPLC-PDA Method for Olmesartan Medoxomil and Metoprolol Succinate-Related Substance.

Objectives: Olmesartan medoxomil (OLM) and metoprolol succinate (MPS) in fixed-dose combination (FDC) tablet formulation prescribed extensively. Stability indicating (SI) method for impurities and related substance (RS) test quantitates the amount of these analytes in formulation; the manuscript presents SI/RS-ultra-high performance liquid chromatography-photodiode array (UHPLC-PDA) method for OLM and MPS and their impurities. Materials and Methods: Well-resolved separation of all analytes was achieved with gradient elution on a Shimadzu on Shimpack GIST-C18 (100 mm x 2.1 mm, 2 µm) column maintained at 25°C. Mobile phase-A consist of 0.1% orthophosphoric acid in water and mobile phase-B was acetonitrile at a flow rate of 0.4 mL/min, data integrated at 225 nm and 16 min of short runtime for satisfactory elution of all peaks. Results: The proposed SI/RS-UHPLC-PDA method was developed and validated as per International Conference on Harmonisation (ICH) of Technical Requirements guidelines. The system suitability test complied by all eluted peaks of the interest with acceptable linearity, recovery, and precision. Specificity, robustness, and method sensitivity parameters were determined; all the parameters were found to be within the limits. All the impurities and stress-degraded peaks were well resolved. Conclusion: The proposed method was found to be simple, fast, linear, and accurate. Further, the method is precise, robust, and specific; suitable for routine IPQC during active pharmaceutical ingredient manufacturing, stability and impurity profiling studies of the titled bulk analytes. Furthermore, the method can be extended to assess the levels of impurities formed during life cycle of new FDCs of titled analytes.

Ultra-High-Performance Liquid Chromatography with Photodiode Array and High-Resolution Time-of-Flight Mass Spectrometry Detectors (UHPLC-PDA-ESI-ToF/HRMS) for the Tentative Structural Characterization of Bioactive Compounds of Salvia verbenaca Extracts in Relation to Their Biological Activities.

BACKGROUND: Salvia verbenaca of the Lamiaceae family is a Mediterranean plant widely used in the Moroccan traditional folk medicine. The aim of this work was to explore the phytochemical composition of Salvia verbenaca extracts and its antioxidant activity. METHODS: Separation and identification of the major phytochemicals present in the two hexane and ethyl acetate explored extracts have been achieved through ultra-high-performance liquid chromatography separation technique coupled to photodiode array and high-resolution time-of-flight mass spectrometry detectors. Antioxidant activity of the obtained extracts was evaluated through DPPH• (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azinobis(3-ethylbenzothiazoline6-sulfonic acid) diammonium salt) assays and the obtained results were compared to TROLOX (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid). RESULTS: Using the analysis technique indicated above, eighteen phytochemicals belonging to phenolic acids, phenolic diterpenes and flavonoids have been characterized on the basis of the obtained UV and mass spectroscopic properties and on the subsequent fragmentations. The antioxidant activity of the explored extracts showed potential scavenging activities compared to TROLOX. A detailed discussion of the attained results has been presented considering the activities observed of each extract. CONCLUSIONS: The research herein presented an analysis technique allowing to screen Salvia verbenaca phytochemicals. The explored plant could be considered as a source of functional phenolic compounds. These could be useful for further pharmacological studies such as new drugs design after clinic and its safety evaluation. It is thus hoped that the information presented here might prompt further studies that will possibly lead to development of therapeutic agents from this plant.

Highly oxidized flavones in Artemisia species - structure revisions and improved UHPLC-MSn analysis.

In course of our studies of the aerial parts of Artemisia abrotanum the major methoxyflavonol could be isolated. However, by NMR structural analysis it became obvious that the substitution pattern in ring B differs from reports for casticin (2). The position of methoxyl and hydroxyl groups are interchanged, i.e., the major flavone is actually chrysosplenetin (1). Three structures in A. abrotanum and A. frigida had to be revised. Use of pyridine-d5 instead of DMSO­d6 made the resolution of the B-ring 1H and 13C NMR signals possible and enabled correct structural assignment by 2D NMR experiments. Results from NMR structure elucidation for A. abrotanum were confirmed by LC-PDA-ESI-MSn analysis when a PFP (pentafluorophenyl) stationary phase with an optimized gradient elution was applied for separation of 1 and 2 instead of a corresponding C-18 phase. Electrospray mass spectrometry (positive and negative mode) with subsequent fragmentation (ESI-MSn) revealed distinctive mass spectral features of both compounds, especially at MS4 level. Several Artemisia extracts including A. annua were analysed on the PFP phase for the presence of 1 and 2.

The Antioxidant and Anti-Inflammatory Properties of Merremia umbellata Extract.

Merremia umbellata Hallier f. (MU) has been used as an anti-inflammatory agent to treat burns and scales. However, the potential anti-inflammatory mechanisms of action of this plant have not been elucidated. This study aimed to assess the antioxidant and anti-inflammatory effects of the leaf and shoot of MU grown in Bangladesh. The MU extract exhibited antioxidant activities as demonstrated by DPPH and ABTS free-radical-scavenging activities and the total polyphenol and total flavonoid contents. MU extract significantly reduced the lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in RAW264.7 macrophage. Accordingly, the gene levels of inducible NO synthase and cyclooxygenase-2 were suppressed. The MU extract alleviated the LPS-induced expression of TLR4, NF-κB, and inflammatory cytokines (TNF-α, IL-6, and IL-1ß). The constituents of a MU extract were tentatively identified using UHPLC-PDA-QTOF/MS techniques. The main compounds were identified as 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, quercitrin, and 4,5-dicaffeoylquinic acid. Molecular docking analysis revealed that these compounds interact with TLR4 protein, with quercitrin showing the highest binding affinity among them. Overall, our findings demonstrate the antioxidant and in vitro anti-inflammatory activities of MU and its potential compounds to target the TLR4-NF-κB signaling pathway. These findings are potentially used to further explore promising natural food ingredients that are effective in regulating inflammation.

High throughput analytical approach based on µQuEChERS combined with UHPLC-PDA for analysis of bioactive secondary metabolites in edible flowers.

Mallow blue (Malva sylvestris L.), hibiscus (Hibiscus rosa-sinensis L.) and nasturtium (Tropaeolum majus L.), are common edible flowers rich in bioactive secondary metabolites (BASMs) whose use in sophisticated gastronomy present currently as increasing trend. In this study the BASMs profile of these edible flowers was established using an emerging green extraction technique, µQuEChERS followed by ultra-high performance liquid chromatography coupled to a photodiode array detection system (UHPLC-PDA). After validation the µQuEChERS/UHPLC-PDA methodology allow to identify that apigenin and epigallocatechin gallate are the most abundant BASMs in mallow blue flowers, while catechin and dicaffeoylquinic acid are predominant in hibiscus flowers, and myricitrin and dicaffeoylquinic acid in nasturtium flowers. Total polyphenol content is the highest in the extract of hibiscus. Nasturtium shows the greatest radical scavenging activity. The results revealed that these flowers constitute a potential source of BASMs with different bioactive properties suggesting its use in design of new functional foods.

UHPLC-PDA-ESI-MS profile of phenolic compounds in the aerial parts of Cuphea ingrata Cham. & Schltdl.

The purpose of the current study was a qualitative UHPLC-PDA-ESI-MS analysis of phenolic compounds in the aerial parts of Cuphea ingrata, which led to detection of over sixty constituents: tannins, flavonoids, phenolic acids and their derivatives. The presence of oenothein B-type macrocyclic dimeric ellagitannins seems to be of particular importance. Quercetin sulfate, that has been previously identified as characteristic chemotaxonomic marker in Cuphea carthagenensis, was found in C. ingrata, as well.

Optimization of Astaxanthin Recovery in the Downstream Process of Haematococcus pluvialis.

Astaxanthin derived from Haematococcus pluvialis is a valuable metabolite applied in a wide range of products. Its extraction depends on a sophisticated series of downstream process steps, including harvesting, disruption, drying, and extraction, of which some are dependent on each other. To determine the processes that yield maximum astaxanthin recovery, bead milling, high-pressure homogenization, and no disruption of H. pluvialis biomass were coupled with spray-drying, vacuum-drying, and freeze-drying in all possible combinations. Eventually, astaxanthin was extracted using supercritical CO2. Optimal conditions for spray-drying were evaluated through the design of experiments and standard least squares regression (feed rate: 5.8 mL/min, spray gas flow: 400 NL/h, inlet temperature: 180 °C). Maximal astaxanthin recoveries were yielded using high-pressure homogenization and lyophilization (85.4%). All combinations of milling or high-pressure homogenization and lyophilization or spray-drying resulted in similar recoveries. Bead milling and spray-drying repeated with a larger spray-dryer resulted in similar astaxanthin recoveries compared with the laboratory scale. Smaller astaxanthin recoveries after the extraction of vacuum-dried biomass were mainly attributed to textural changes. Evaluation of these results in an economic context led to a recommendation for bead milling and spray-drying prior to supercritical CO2 extraction to achieve the maximum astaxanthin recoveries.