Liquid Chromatography-electrospray Mass Spectrometry Research Articles

Mitochondrial Disruption by Amyloid Beta 42 Identified by Proteomics and Pathway Mapping.

Alzheimer’s disease (AD) is marked by chronic neurodegeneration associated with the occurrence of plaques containing amyloid β (Aβ) proteins in various parts of the human brain. An increase in several Aβ fragments is well documented in patients with AD and anti-amyloid targeting is an emerging area of therapy. Soluble Aβ can bind to various cell surface and intracellular molecules with the pathogenic Aβ42 fragment leading to neurotoxicity. Here we examined the effect of Aβ42 on network adaptations in the proteome of nerve growth factor (NGF) differentiated PC12 cells using liquid-chromatography electrospray ionization mass spectrometry (LC-ESI MS/MS) proteomics. Whole-cell peptide mass fingerprinting was coupled to bioinformatic gene set enrichment analysis (GSEA) in order to identify differentially represented proteins and related gene ontology (GO) pathways within Aβ42 treated cells. Our results underscore a role for Aβ42 in disrupting proteome responses for signaling, bioenergetics, and morphology in mitochondria. These findings highlight the specific components of the mitochondrial response during Aβ42 neurotoxicity and suggest several new biomarkers for detection and surveillance of amyloid disease.

Anion-Responsive Manganese Porphyrin Facilitates Chloride Transport and Induces Immunogenic Cell Death

Anion-Responsive Manganese Porphyrin Facilitates Chloride Transport and Induces Immunogenic Cell Death

Identification of pesticides in water samples by solid-phase extraction and liquid chromatography-electrospray ionization mass spectrometry.

The Contaminants of Emerging Concern (CECs), including pesticides, have been a trending topic and Brazil is the country with the highest usage of pesticides worldwide. This study aimed to measure the presence of pesticide residues in the water from different sources in the city of Porto Alegre. We analyzed 55 samples from drinking water treatment plants, public water sites, and sewage treatment plants from winter 2018 to summer 2020 by solid-phase extraction and high-performance liquid chromatography-electrospray ionization mass spectrometry. Among 184 pesticides evaluated, 107 matched validation criteria (linearity, trueness, accuracy, repeatability, reproducibility) and 15 of them were detected in different water samples, including seven insecticides, five antifungals, and three herbicides, with a wide range of toxicity levels and noticeable seasonal differences. For the worst-case scenario evaluation, 20 out of 22 (90.9%) samples exceeded the Risk Quotient of 1. The sum of pesticide concentrations exceeded 100 ng L-1 in 66.7% of samples in February 19 and in 75% of samples in February 20 and the total pesticide concentration has reached the worrisome mark of 1615 and 954.96 ng L-1 respectively. Therefore, our results make evident the need to promote public policies to achieve better water quality monitoring. PRACTITIONER POINTS: Among 184 pesticides evaluated, 107 matched validation criteria (linearity, trueness, accuracy, repeatability, reproducibility). A total of 55 different water samples were analyzed, and 15 pesticides were detected and five quantified. For the worst-case scenario evaluation, 20 out of 21 samples exceeded the Risk Quotient of 1 on Feb/20. The pesticide concentrations sum exceeded 100 ng L-1 in 66.7% of samples on February 19 and in 75% of samples on February 20. It is mandatory to improve water monitoring to guide the development of public policies concerning its quality.

Coelomic fluid of Echinometra mathaei: The new prospects for medicinal antioxidants

Echinoid pigments have various biological properties such as antioxidant, cytotoxic, and antibacterial activities. We aimed to evaluate the extraction of cell-free coelomic fluid (CFCF) and coelomocyte lysate (CL) as well as qualitatively and quantitatively identify the coelomic fluid of Echinometra mathaei as a new source of polyhydroxylatednaphthoquinone (PHNQ) antioxidant pigments. Based on the High Performance liquid chromatography–electrospray mass spectrometry (HPLC-MS) analysis in negative mode, the main quinonoid (PHNQ) pigments were identified and quantified. This study also illustrated the total ion current chromatograms and related mass spectra of Spinochrome A, Spinochrome B, Spinochrome C, and Echinochrome A in CL and SpinochromeC in CFCF samples. The ions at 221, 279, 265 and 263 m/z correspond to the pseudo-molecular [M − H] ions of Spinochrome B, Spinochrome C, Echinochrome A, and Spinochrome A, respectively. These components have previously been noted from the shells and spines of sea urchins but identification of PHNQs pigments in CL and CFCF of E. mathaei using LC-MS was introduced for the first time. The results also showed that, the highest DPPH radical scavenging activity of CFCF (88.12 DPPH% scavenging at 70 μg/mL, IC50 = <10 μg/mL). The findings clearly suggest that the coelomic fluid of E. mathaei could be served as the promising as well as potential natural antioxidants in the medical and pharmaceutical industries and could replace the increasing prices of the commercial antioxidants products.

Organic complexation of iron by strong ligands and siderophores in the eastern tropical North Pacific oxygen deficient zone

Continental margins are an important external source of dissolved iron to the marine environment. However, the mechanisms responsible for the offshore transport of dissolved iron is impacted by the resulting iron speciation. We characterized the iron speciation in the Eastern Tropical North Pacific (ETNP) oxygen deficient zone (ODZ), including dissolved iron, organic iron-binding ligands, and reduced iron. Organic iron-binding ligands were measured using both competitive ligand exchange adsorptive cathodic stripping voltammetry (CLE-ACSV) and liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) in order to explore the impact of organic ligands on dissolved iron (dFeT) and iron(II) biogeochemistry in the region. Organic ligands were present in high concentrations (1.06–5.30 nmol L−1) and exceeded dissolved iron concentrations (0.36–4.52 nmol L−1) at all locations. Iron-binding strengths (logKFeL,Fe′cond) ranged 11.22 to 12.75 and were elevated in the ODZ layer relative to the oxygenated water column. LC-ESI-MS revealed the presence of siderophores, or bacterially-produced organic ligands with high Fe-affinity, in all samples analyzed, suggesting these compounds may be produced by microbes in the ODZ despite high ambient dFeT concentrations. This study is the first to characterize siderophores in an ODZ environment to date, and the three siderophores found (amphibactin B, synechobactin c9, synechobactin c10) could contribute to the observed elevated logKFeL,Fe′cond of ligands in the ODZ. Comparative analysis of organic ligand logKFeL,Fe′cond values in other low oxygen environments suggests that strong ligands, including siderophores, could be present in other low oxygen regions. In a simple model of the shelf-to-offshore iron transport mechanism, strong organic iron-binding ligands had a large impact on the longevity and transport of iron in the ODZ. These results suggest that organic ligand composition can have an impact on iron distributions in the ETNP ODZ and regulate the offshore transport of iron to the open ocean.

Secondary metabolic profiles and anticancer actions from fruit extracts of immature pomegranates.

Immature fruits from Punica granatum L. thinning are a neglected side product of pomegranate production with cumbersome disposal costs for farmers. To explore value potential of immature fruits from pomegranate ‘Wonderful’ cultivars, the compositional landscapes and antitumorigenic activities of pomegranate extracts from two different stages of maturation were assessed. Cancer cell proliferation and cytotoxicity was quantified in human lung H1299 and colon HCT116 adenocarcinomas by crystal violet staining, MTS assay and caspase-3 activity. High performance liquid chromatography—diode array detector (HPLC/DAD) and high performance liquid chromatography—electrospray ionization—mass spectrometry (HPLC/ESI-MS) analyses indicate that immature fruits are rich sources of gallotannins and ellagitannins, with the highest amounts contained in immature fruit peels. Biological investigations reveal a robust anticancer activity by those immature P. granatum fruit extracts, which reflected induction of tumor cytotoxicity and cell death mechanisms. Together, present observations suggest P. granatum byproducts from the thinning process may provide unexplored values for virtuous circular economy.

Improved identification and quantification of peptides in mass spectrometry data via chemical and random additive noise elimination (CRANE).

MotivationThe output of electrospray ionization–liquid chromatography mass spectrometry (ESI-LC-MS) is influenced by multiple sources of noise and major contributors can be broadly categorized as baseline, random and chemical noise. Noise has a negative impact on the identification and quantification of peptides, which influences the reliability and reproducibility of MS-based proteomics data. Most attempts at denoising have been made on either spectra or chromatograms independently, thus, important 2D information is lost because the mass-to-charge ratio and retention time dimensions are not considered jointly.ResultsThis article presents a novel technique for denoising raw ESI-LC-MS data via 2D undecimated wavelet transform, which is applied to proteomics data acquired by data-independent acquisition MS (DIA-MS). We demonstrate that denoising DIA-MS data results in the improvement of peptide identification and quantification in complex biological samples.Availability and implementationThe software is available on Github (https://github.com/CMRI-ProCan/CRANE). The datasets were obtained from ProteomeXchange (Identifiers—PXD002952 and PXD008651). Preliminary data and intermediate files are available via ProteomeXchange (Identifiers—PXD020529 and PXD025103).Supplementary information Supplementary data are available at Bioinformatics online.

Distribution analysis of jasmonic acid-related compounds in developing Glycine max L. (soybean) seeds using mass spectrometry imaging and liquid chromatography-mass spectrometry.

Jasmonic acid (JA) and its precursors are oxylipins derived from α-linolenic acid (αLA) and hexadecatrienoic acid, and regulate seed development. However, their spatial distribution in the developing Glycine max L. (soybean) seeds has not been elucidated. To investigate the distribution of JA-related compounds in the developing soybean seeds using desorption electrospray ionisation-mass spectrometry imaging (DESI-MSI) and liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) analyses. Cryosections of developing seeds were prepared using adhesive films, and subjected to DESI-MSI analysis. Verification of the DESI-MSI ion images were performed using DESI-tandem MSI (MS/MSI), LC-ESI-MS and tandem MS (MS/MS). In the DESI-MSI mass spectrum, peaks matching the chemical formulae of αLA, 12-oxo-phytodienoic acid (OPDA), and 3-oxo-2-(2-(Z)-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0) were detected. These compounds were mainly distributed in the seed coat, especially near the hilum. This was consistent with the quantitative results obtained by LC-ESI-MS. While, DESI-MS/MSI and LC-ESI-MS/MS suggested the presence of isomers for OPDA and OPC-8:0. The effect of isomers on the DESI-MSI ion images was small for OPDA, and considerable for OPC-8:0. These results demonstrated that free αLA, OPDA, and OPC-8:0 were the abundant JA-related compounds mainly distributed in the seed coat of the developing soybeans. OPDA and OPC-8:0 might exert a biological role in the seed coat. To the best of my knowledge, this is the first report on the accumulation of OPDA and OPC-8:0 in the seed coat. The combination of DESI-MSI and LC-ESI-MS is a useful tool for distribution analysis of JA-related compounds in the developing seeds.

Revisiting N-glycosylation in Halobacterium salinarum: Characterizing a dolichol phosphate- and glycoprotein-bound tetrasaccharide.

Although Halobacterium salinarum provided the first example of N-glycosylation outside the Eukarya, much regarding such post-translational modification in this halophilic archaea remains either unclear or unknown. The composition of an N-linked glycan decorating both the S-layer glycoprotein and archaellins offers one such example. Originally described some 40years ago, reports from that time on have presented conflicted findings regarding the composition of this glycan, as well as differences between the protein-bound glycan and that version of the glycan attached to the lipid upon which it is assembled. To clarify these points, liquid chromatography-electrospray ionization mass spectrometry was employed here to revisit the composition of this glycan both when attached to selected asparagine residues of target proteins and when bound to the lipid dolichol phosphate upon which the glycan is assembled. Such efforts revealed the N-linked glycan as corresponding to a tetrasaccharide comprising a hexose, a sulfated hexuronic acid, a hexuronic acid and a second sulfated hexuronic acid. When attached to dolichol phosphate but not to proteins, the same tetrasaccharide is methylated on the final sugar. Moreover, in the absence of the oligosaccharyltransferase AglB, there is an accumulation of the dolichol phosphate-linked methylated and disulfated tetrasaccharide. Knowing the composition of this glycan at both the lipid- and protein-bound stages, together with the availability of gene deletion approaches for manipulating Hbt. salinarum, will allow delineation of the N-glycosylation pathway in this organism.

Recent Advances in Mass Spectrometry-Based Glycomic and Glycoproteomic Studies of Pancreatic Diseases.

Modification of proteins by glycans plays a crucial role in mediating biological functions in both healthy and diseased states. Mass spectrometry (MS) has emerged as the most powerful tool for glycomic and glycoproteomic analyses advancing knowledge of many diseases. Such diseases include those of the pancreas which affect millions of people each year. In this review, recent advances in pancreatic disease research facilitated by MS-based glycomic and glycoproteomic studies will be examined with a focus on diabetes and pancreatic cancer. The last decade, and especially the last five years, has witnessed developments in both discovering new glycan or glycoprotein biomarkers and analyzing the links between glycans and disease pathology through MS-based studies. The strength of MS lies in the specificity and sensitivity of liquid chromatography-electrospray ionization MS for measuring a wide range of biomolecules from limited sample amounts from many sample types, greatly enhancing and accelerating the biomarker discovery process. Furthermore, imaging MS of glycans enabled by matrix-assisted laser desorption/ionization has proven useful in complementing histology and immunohistochemistry to monitor pancreatic disease progression. Advances in biological understanding and analytical techniques, as well as challenges and future directions for the field, will be discussed.

Bioactive Molecules for Discriminating Robinia and Helianthus Honey: High-Performance Liquid Chromatography-Electron Spray Ionization-Mass Spectrometry Polyphenolic Profile and Physicochemical Determinations.

Bioactive molecules from the class of polyphenols are secondary metabolites from plants. They are present in honey from nectar and pollen of flowers from where honeybees collect the “raw material” to produce honey. Robinia pseudoacacia and Helianthus annuus are important sources of nectar for production of two monofloral honeys with specific characteristics and important biological activity. A high-performance liquid chromatography–electro spray ionization–mass spectrometry (HPLC–ESI–MS) separation method was used to determine polyphenolic profile from the two types of Romanian unifloral honeys. Robinia and Helianthus honey showed a common flavonoid profile, where pinobanksin (1.61 and 1.94 mg/kg), pinocembrin (0.97 and 1.78 mg/kg) and chrysin (0.96 and 1.08 mg/kg) were identified in both honey types; a characteristic flavonoid profile in which acacetin (1.20 mg/kg), specific only for Robinia honey, was shown; and quercetin (1.85 mg/kg), luteolin (21.03 mg/kg), kaempferol (0.96 mg/kg) and galangin (1.89 mg/kg), specific for Helianthus honey, were shown. In addition, different phenolic acids were found in Robinia and Helianthus honey, while abscisic acid was found only in Robinia honey. Abscisic acid was correlated with geographical location; the samples collected from the south part of Romania had higher amounts, due to climatic conditions. Acacetin was proposed as a biochemical marker for Romanian Robinia honey and quercetin for Helianthus honey.

Low-polymerization compositional fingerprinting for characterization of Schisandra polysaccharides by hydrophilic interaction liquid chromatography-electrospray mass spectrometry

A hydrophilic interaction liquid chromatography-negative electrospray-mass spectrometry (HILIC-ESI−-MS) coupled with microwave assisted mild acid (MAMA) depolymerization is proposed here for unusual discrimination and characterization of plant polysaccharides: a case study of fruit polysaccharides in Schisandra chinensis and S. sphenanthera (SCP and SSP). The optimized MAMA hydrolysis procedure was proposed for sample preparations of low-polymerization saccharides (Mw < 5000 Da) released in SCP and SSP. In addition, HILIC-MS/MS was employed for elucidation of isomeric glycosidic linkages in terms of 18O labelling. The MAMA hydrolysates showed that the amount of neutral →(4Hex1)n→ moiety is confirmed to be more bigger than that of acidic →(4HexA1)n → in SCP, whereas the amount of acidic →(4HexA1)n→ moiety seems to be more bigger than that of neutral →(4Hex1)n→ in SSP. The resulting low-polymerization compositional fingerprinting (LCF) showed the performance on rapid visualization of SCP and SSP by HILIC-MIM-MS. Principal components analysis (PCA) and hierarchical cluster analysis (HCA) further unveils several key Q-markers (e.g., m/z 503, 369, 665, 827, 989, 1151 and 735) for rapid discrimination of SCP and SSP. This practical study showed that the LCF with PCA and HCA could effectively reflect structural differences and could rapidly achieve discrimination of SCP and SSP.

Spectral Characteristics Related to Chemical Substructures and Structures Indicative of Organic Precursors from Fulvic Acids in Sediments by NMR and HPLC-ESI-MS.

The aim of this work was to determine Fulvic Acids (FAs) in sediments to better know their composition at the molecular level and to propose substructures and structures of organic precursors. The sediment samples were obtained from a priority area for the conservation of ecosystems and biodiversity in Mexico. FAs were extracted and purified using modifications to the International Humic Substances Society method. The characterization was carried out by 1D and 2D nuclear magnetic resonance (NMR) and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) in positive (ESI+) and negative (ESI−) modes. Twelve substructures were proposed by the COSY and HSQC experiments, correlating with compounds likely belonging to lignin derivatives obtained from soils as previously reported. The analysis of spectra obtained by HPLC-ESI-MS indicated likely presence of compounds chemically similar to that of the substructures elucidated by NMR. FAs studied are mainly constituted by carboxylic acids, hydroxyl, esters, vinyls, aliphatics, substituted aromatic rings, and amines, presenting structures related to organic precursors, such as lignin derivatives and polysaccharides.

Determination of SK7324, a new class of hepatitis C virus inhibitor, in rat plasma by UPLC-MS/MS and its application to the pharmacokinetic study of SK7324.

In this study, a sensitive method for quantitation of the unique small-molecule inhibitor of hepatitis C virus SK7324 in rat plasma has been established using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI/MS/MS). SK7324 and internal standard (tramadol) in plasma sample was extracted using acetonitrile. A centrifuged upper layer was then evaporated and reconstituted with the mobile phase of 0.5% formic acid-acetonitrile (35:65, v/v). The reconstituted samples were injected into a C18 reversed-phase column. Using MS/MS in the multiple reaction monitoring (MRM) mode, SK7324 and tramadol were detected without severe interference from rat plasma matrix. Detection of SK7324 in rat plasma by the UPLC-ESI/MS/MS method was accurate and precise with a quantitation limit of 1.0 ng/mL. The validation, reproducibility, stability, and recovery of the method were evaluated. The method has been successfully applied to pharmacokinetic studies of SK7324 in rat plasma. Pharmacokinetic parameters of SK7324 were evaluated after intravenous (i.v.; at doses of 5 mg/kg) and oral (p.o.; at doses of 10 mg/kg) administration of SK7324 in rats. After p.o. administration (10 mg/kg) of SK7324, F (Fraction absorbed) value was approximately 87.1%.

Separation, kinetics of purification of syringin by macroporous resin from Syringa oblata Lindl. leaves and the evaluation of bioactivities

PurposeThis paper aims to explore the adsorption kinetics of syringin from Syringa oblata Lindl. leaves on macroporous resin and develop an efficient, simple and recyclable technology for the separation and purification of syringin.Design/methodology/approachStatic adsorption and desorption properties of six resins were tested to select a suitable resin for the purification of syringin. Langmuir and Freundlich isotherm models were used to estimate the adsorption behavior of syringin on AB-8 resin. Breakthrough point and eluent volume were determined by dynamic adsorption and desorption tests. High-performance liquid chromatography-electrospray ionization-mass spectrometry was applied to identify the syringin in the purified product [syringin product (SP)]. Antioxidant and antibacterial activities of SP in vitro were evaluated by free radical scavenging ability and biofilm formation inhibitory tests.FindingsAB-8 exhibited the most suitable adsorption and desorption capacity. Adsorption isotherm parameters indicated favorable adsorption between AB-8 and syringin. The optimal results were as follows: for adsorption, the sample concentration was 1.85 mg/mL, the sample volume was 3.5 bed volume (BV), the flow rate was 0.5 mL/min; for desorption, the ethanol concentration was 70%, the elution volume was 2.5 BV, the elution velocity was 1.0 mL/min. SP with 80.28% syringin displayed the potent antioxidant activities and inhibitory effects on biofilm formation of Streptococcus suis.Originality/valueTo the best of authors’ knowledge, there are no reports on purifying syringin from Syringa oblata Lindl. leaves using macroporous resins. This paper may also provide a theoretical reference for the purification of other phenylpropanoid glucosides.

Quantification of bovine plasma amino acids via liquid chromatography–electrospray ionization-mass spectrometry: Comparison of underivatized and precolumn derivatized methods

The objectives of this experiment were to evaluate and compare underivatized (UND) and precolumn derivatized (DER) methods for quantification of bovine plasma AA by isotope dilution ratio via liquid chromatography-electrospray ionization (ESI)-single quadrupole mass spectrometry. Linearity of the mass-to-charge ratio signal and area signal sensitivity of 12C were evaluated for each AA with 5-point standard curves (range: 1.1–500 µM). Plasma from lactating dairy cows was isolated by centrifugation and deproteinized using 1 N perchloric acid with a final concentration of 0.5 N. Deproteinized plasma was filtered and injected into a 50 × 2-mm column (Imtakt) or extracted, derivatized, and injected into a 250 × 3-mm column (EZ:faast, Phenomenex) and analyzed via liquid chromatography-ESI-single quadrupole mass spectrometry. Coefficients of variation and recovery rates were evaluated using 4 replicates of pooled plasma samples spiked with each AA at concentrations of 10, 20, and 50 µM. In addition, a subset of 24 plasma samples was used to directly compare methods using linear regression, correlation coefficient (r), concordance correlation coefficient (CCC), and Bland-Altman plot test. Both methods showed linearity within the dynamic range analyzed for all essential AA (coefficient of determination, R2 ≥ 0.995) and most other AA, although the UND samples had poor linearity (R2 ≤ 0.990) or peak resolution problems for Asp, Gly, Tyr, and Ser. Moreover, area signal sensitivity for 12C AA was greater for DER samples than for UND samples [range: 2.2× (Pro) to 309.5× (Ala)]. Both methods had recovery rates ranging from 85.7 to 119.8.0%, and none differed from 100% except Gln [20 µM (85.7%) and 50 µM (87.6%)] and Val [50 µM (119.8%)] using the UND method. The UND method had a coefficient of variation ranging from 0.9% (Val) to 7.8% (His), whereas for the DER method the range was 2.2% (Glu) to 8.8% (Asp). The highest correlation coefficient (>0.90) and CCC (>0.90) were observed for Arg, Ile, Leu, Met, Thr, Trp, Val, and Gln, with the Bland-Altman plot test showing minimal mean bias for these AA. Lowest values were observed for His (r = 0.46; CCC = 0.45), Lys (r = 0.76; CCC = 0.75), Ala (r = 0.83; CCC = 0.73), and Glu (r = 0.65; CCC = 0.42). The UND method showed linearity, precision, and accurate recovery rates for most AA, with most essential AA having comparable values between the 2 methods. However, the DER method had greater 12C AA area signal sensitivity, linearity, and recovery rates.

Simultaneous extraction of polysaccharides and polyphenols from blackcurrant fruits: Comparison between response surface methodology and artificial neural networks

A procedure for simultaneous extraction of polysaccharides and polyphenols from blackcurrant fruits by microwave-assisted aqueous two-phase system (MA-ATPS) was studied. Ethanol/(NH4)2SO4 was determined as the most suitable ATPS including 18.99 % (NH4)2SO4 and 31.65 % of absolute ethanol. Apart from response surface methodology (RSM), artificial neural network linked genetic algorithms and grey relational analysis (ANN-GA-GRA) was also employed to optimize the extraction process of polysaccharides and polyphenols from blackcurrant fruits. GA was used to give assistance to the parameters optimization of ANN model and GRA was subsequently utilized to optimize the variables. ANN model displayed relatively higher coefficient of determination (R2, 0.9805) and lower absolute average deviation (AAD, 0.6648) than those of RSM (R2 = 0.9783, AAD = 0.8505), indicating ANN possessed more excellent prediction and estimation capabilities. Under the optimum conditions (liquid-to-material ratio, extraction time, extraction temperature and microwave power predicted by RSM were 60.01 mL/g, 9.96 min, 59.57 °C, and 592.98 W, while predicted by ANN were 56.10 mL/g, 9.78 min, 60.92 °C, and 640.78 W), the yields of polysaccharides, anthocyanins, polyphenols, and DPPH radical scavenging activities of two phases given by ANN (46.75 %, 6.75 mg/g, 53.51 mg/g, 95.17 %, and 97.05 %) were higher than those deriving from RSM (43.48 %, 6.62 mg/g, 52.48 mg/g, 93.72 %, and 96.65 %). In addition, gas chromatography (GC) analysis confirmed that the polysaccharides in bottom phase were composed of six monosaccharides, and thirteen phenolic compounds were determined in the extract of top phase by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). Therefore, microwave-assisted ethanol/(NH4)2SO4 extraction may be a promising technique to simultaneously prepare multiple active compounds from blackcurrant, and ANN-GA-GRA was an effective mathematical model to predict and optimize this extraction process.

Antihypernociceptive effects of Petersianthus macrocarpus stem bark on neuropathic pain induced by chronic constriction injury in rats.

Petersianthus macrocarpus (Lecythidaceae) stem bark is traditionally used in West and Central Africa for the treatment of boils and pain. The present study examined the chemical composition of the aqueous and methanolic stem bark extracts of P. macrocarpus by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) . Their antinociceptive effect was evaluated using chronic constriction injury (CCI)-induced neuropathic pain in a rat model. On the ninth day post-surgery, the pain perception (allodynia and hyperalgesia) of the animals was assessed after the administration of aqueous and methanolic extracts at the doses of 100 and 200mg/kg. In addition, the effect of the extracts was evaluated on nitric oxide activity and on the expression of pro-inflammatory cytokines (TNF-α, IL-1β, and NF-κB). The LC-ESI-MS analysis revealed the presence of ellagic acid as the major constituent in the methanol extract. Both extracts at the employed doses (100 and 200mg/kg), significantly (p<0.01 and p<0.001) reduced the spontaneous pain, tactile and cold allodynia, and mechanical hyperalgesia. The methanolic extract used at the dose of 200mg/kg significantly reduced the nitric oxide level (p<0.001) and the gene expression levels of NF-κB (p<0.05) and TNF-α (p<0.01) in the brain. These data may indicate that stem bark extracts of P. macrocarpus possess a potent anti-hypernociceptive effect on CCI neuropathic pain. The inhibition of the nitric oxide pathway as well as the reduction in NF-κB and TNF-α gene expression in the brain may at least partially contribute to this effect. The results further support the use of this plant by traditional healers in pain conditions.

Vaccinium bracteatum Thunb. fruit extract reduces high-fat diet-induced obesity with modulation of the gut microbiota in obese mice.

Vaccinium bracteatum Thunb. fruits have been used as traditional food. This study investigated the effects of a polyphenol-rich Vaccinium bracteatum Thunb. fruit extract (VBTE) on obesity and obesity-related diseases in mice, and the potential role of the gut microbiota in the bioactivity of VBTE was also determined. Chemical constituents of the VBTE were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS). C57BL/6J mice (weighing 17.8-21.6g) were fed a low-fat diet (LFD) or high-fat diet (HFD) with or without VBTE treatment for 14weeks. The gut microbial changes were determined using 16S rRNA sequencing. Our results showed that VBTE mainly contains 36 kinds of polyphenols. VBTE reduced HFD-induced body weight gain by 33.42% (p<.05), steatosis scores by 56.25% (p<.05), and insulin resistance index by 51.49% (p<.05). Moreover, VBTE altered the composition of the gut microbiota. The correlation analysis indicated that Akkermansia, Alistipes, Bacteroides, Alloprevotella, Ruminiclostridium, Ruminiclostridium_9, and Rikenellaceae_RC9_gut_group were negatively correlated with serum lipids, glucose, and insulin, while Escherichia-Shigella was positively associated with these clinical indicators. In conclusion, VBTE supplement could reduce obesity and be a treatment option for obesity-related diseases by influencing the gut microbiota in mice. PRACTICAL APPLICATIONS: Plant extracts are widely used to treat obesity and related metabolic disorders. Polyphenols, the well-known natural antioxidants present in fruits, are consumed as a dietary supplement to prevent many diseases. Recent pharmacological studies have reported that Vaccinium bracteatum Thunb. fruits have many physiological functions, such as anti-proliferative, anti-inflammatory, and antidepressant-like effects. Despite these properties of Vaccinium bracteatum Thunb. fruits, their anti-obesity effect has not been studied to date. The findings of this study will support VBTE could be used as an important therapeutic application for preventing obesity and related metabolic diseases by modulating the gut microbiota.

Improving sesquiterpenoids production of Sarcandra glabra callus culture

Sarcandra glabra, a traditional Chinese medicine and herbal tea, has benefits from treating inflammation and fractures to curing cancer. The anti-inflammatory properties of sesquiterpenoids (chloranthalactone A, CTA; atractylenolide II, AT-II) have made S. glabra to a great potential for research and development. Here this study developed S. glabra callus culture to circumvent particular needs of the plant growing seasons and Hardiness zone, with the goal being able to produce sesquiterpenoids in vitro. The half strength Murashige and Skoog (1/2MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 2.0 mg/L) and 6-benzylaminopurine (6-BA, 1.0 mg/L) was effective for callus induction. The 1/2MS medium supplemented with 2,4-D (2.0 mg/L) and 6-BA (0.5 mg/L) was superior for callus proliferation. The callus was friable, yellowish and rapidly grown without tissue differentiation. Moreover, high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis indicated that the contents of CTA and AT-Ⅱ in multiple subculture S. glabra callus were 3.72 ± 0.51 mg/g and 0.50 ± 0.12 mg/g in fresh weight (FW), respectively. Using abiotic stresses and elicitors, production of CTA (by ultraviolet-B radiation for 48 h) and AT-II (by methyl jasmonate, 100 μmol/L) was enhanced by 2.3-fold. In conclusion, this work not only established the S. glabra callus culture for in vitro production of CTA and AT-II, but also provided the foundation for further development and utilization of S. glabra.