Liquid Chromatography-electrospray Mass Spectrometry Research Articles

Spatial distribution of differential metabolites in different parts of Tetrastigma hemsleyanum Diels et Gilg by ultrahigh-performance liquid chromatography/mass spectrometry and desorption electrospray ionization mass spectrometry imaging

Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) is a homology medicine and food that is rich in bioactive ingredients such as flavonoids and phenolic acids. A strategy that integrates ultrahigh-performance liquid chromatography–electrospray ionization mass spectrometry (UHPLC–ESI/MS) and desorption electrospray ionization mass spectrometry imaging (DESI-MSI) spatial metabolomics analysis was proposed and applied to different parts of T. hemsleyanum for the first time. A total of 132 compounds were tentatively identified, and 3, 7, and 24 components were found only in tuberous roots (TRs), fibrous roots (FRs), and leaves, respectively, using UHPLC–ESI/MS. The DESI-MSI results showed that the spatial distribution of flavonoids between the aerial and underground parts was diverse; for example, vitexin-2″-O-glucoside was largely localized in the epidermis and xylem of TRs, while a small amount was restricted to the xylem of the leaf margin and FRs. There was a higher abundance of most organic acids and flavonoids in the leaves than in the underground parts. Some flavonoids showed the same abundance in FRs and TRs, but the levels of most compounds were considerably higher in FRs. It is possible for leaves and FRs to become new medicinal parts of T. hemsleyanum. Through chemometrics statistics analysis, 19 key differential compounds in different parts of T. hemsleyanum were found, and the pharmacological mechanisms of the main differential metabolites were predicted by network pharmacology. This study provides a novel method for the medicinal development of T. hemsleyanum, showing its great potential in the development of medicinal resources.

LC and LC-MS/MS Studies for the Separation and Identification of Impurities and Degradation Products of Betaxolol.

Betaxolol (Bx) is a selective β1 receptor blocker used in the treatment of hypertension and glaucoma. The aim of the present work was to demonstrate an approach involving use of liquid chromatography (LC) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) for the simultaneous separation, identification and characterization of impurities and of degradation products of betaxolol without their isolation from the reaction mixtures. At optimum condition, and according to ICH guidelines, the limit of detection (LOD) and limit of quantification (LOQ) for Bx are found to be 5.46 and 16.54μgmL-1, respectively. However, the LOD and LOQ for the major degradation product P6 were 2.15 and 6.53μgmL-1. Betaxolol was subjected to hydrolytic (acidic and basic) and oxidative, stress conditions according to International Conference on Harmonization (ICH) guideline Q1A (R2), and as results, the drug was found to be labile in acidic, basic and oxidative stress conditions. Based on LC-ESI/MS analysis, the found results revealed that Bx decomposes in acidic, basic and oxidizing environments. All degradation products were identified with the help of their fragmentation pattern and the masses obtained upon the MS analysis.

Ion-pair chromatographic attribute in the retention of ammonium ion on a crown ether-based and a polysaccharide-based chiral stationary phases. Some mobile phase additives compatible with liquid chromatography-electrospray ionization-mass spectrometry

The design of the mobile phase additive useful in HPLC analysis on two types of chiral stationary phases, one based on chiral crown ether, and another based on polysaccharide derivative, was developed. CROWNPAK® CR-I, the chiral stationary phase bearing (R)- or (S)-(3,30-diphenyl-1,10-binaphtyl)-20-crown-6 (1) moiety is a useful CSP to separate primary amine enantiomers. While perchloric acid had been recommended as the mobile phase additive to attain a better separation, we have reexamined the effect of mobile phase additives and revealed that the retention of an ammonium ion is enhanced by a chaotropic or a hydrophobic anion in a water-based mobile phase like well-known ion pair chromatography in an RPLC. However, the retention was enhanced in reverse by a rather cosmotropic or a hydrophilic anion in an acetonitrile (ACN)-based mobile phase. These results looking contradictory to each other may be rationalized both in the basis of ion pair chromatography. A similar but smaller effect of pairing anion species was also observed on a polysaccharide-based CSP. Based on these findings, some types of mobile phase have been proposed and some of them were demonstrated to be compatible with liquid chromatography-electrospray ionization-mass spectrometry (ESI LC/MS).

Quantification of the aromatase inhibitor letrozole and its carbinol metabolite in mouse plasma by UHPLC-MS/MS

A liquid chromatography - electrospray ionization-mass spectrometry (LC-ESI-MS) method was developed for the quantification of letrozole, a third-generation aromatase inhibitor, and its main carbinol metabolite (CM) in support of murine pharmacokinetic studies. Using polarity switching, simultaneous ESI-MS measurement of letrozole and CM was achieved in positive and negative mode, respectively. The assay procedure involved a one-step protein precipitation and extraction of all analytes from mouse plasma requiring only 5 μL of sample. Separation was optimized on an Accucore aQ column with gradient elution at a flow rate of 0.4 mL/min in 5 min. Two calibration curves per day over four consecutive measurement days showed satisfactory linear responses (r2 > 0.99) over concentration ranges of 5–1000 ng/mL and 20–2000 ng/mL for letrozole and CM, respectively. No matrix effect was found, and the mean extraction recoveries were 103–108 % for letrozole and 99.8–107 % for CM. Precision and accuracy within a single run and over four consecutive measurement days were verified to be within acceptable limits. Application of the developed method to preclinical pharmacokinetic studies in mice receiving oral letrozole at a dose 1 or 10 mg/kg revealed that the systemic exposure to letrozole was dose-, formulation-, and strain-dependent. These findings may inform the future design of preclinical studies aimed at refining the pharmacological profile of this clinically important drug.

Lipidome characterisation and sex-specific differences in type 1 and type 2 diabetes mellitus

BackgroundIn this study, we evaluated the lipidome alterations caused by type 1 diabetes (T1D) and type 2 diabetes (T2D), by determining lipids significantly associated with diabetes overall and in both sexes, and lipids associated with the glycaemic state.MethodsAn untargeted lipidomic analysis was performed to measure the lipid profiles of 360 subjects (91 T1D, 91 T2D, 74 with prediabetes and 104 controls (CT)) without cardiovascular and/or chronic kidney disease. Ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS) was conducted in two ion modes (positive and negative). We used multiple linear regression models to (1) assess the association between each lipid feature and each condition, (2) determine sex-specific differences related to diabetes, and (3) identify lipids associated with the glycaemic state by considering the prediabetes stage. The models were adjusted by sex, age, hypertension, dyslipidaemia, body mass index, glucose, smoking, systolic blood pressure, triglycerides, HDL cholesterol, LDL cholesterol, alternate Mediterranean diet score (aMED) and estimated glomerular filtration rate (eGFR); diabetes duration and glycated haemoglobin (HbA1c) were also included in the comparison between T1D and T2D.ResultsA total of 54 unique lipid subspecies from 15 unique lipid classes were annotated. Lysophosphatidylcholines (LPC) and ceramides (Cer) showed opposite effects in subjects with T1D and subjects with T2D, LPCs being mainly up-regulated in T1D and down-regulated in T2D, and Cer being up-regulated in T2D and down-regulated in T1D. Also, Phosphatidylcholines were clearly down-regulated in subjects with T1D. Regarding sex-specific differences, ceramides and phosphatidylcholines exhibited important diabetes-associated differences due to sex. Concerning the glycaemic state, we found a gradual increase of a panel of 1-deoxyceramides from normoglycemia to prediabetes to T2D.ConclusionsOur findings revealed an extensive disruption of lipid metabolism in both T1D and T2D. Additionally, we found sex-specific lipidome changes associated with diabetes, and lipids associated with the glycaemic state that can be linked to previously described molecular mechanisms in diabetes.

Distribution of Vicine, Convicine and Levodopa in Faba Bean Plant Tissues Determined by UltraHigh Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry

Faba bean is an emerging pulse crop for the Canada Prairies and is becoming an increasingly important source of plant sourced protein. It also has a high content of levodopa (up to 0.1 % of the dry seed weight), a nutraceutical compound for treating Parkinson's Disease (PD). The presence of pyrimidine glycosides, vicine and convicine, however, restricts the use of faba due to their potential to cause favism in susceptible individuals. For the rapid separation and quantification of levodopa, vicine and convicine, a 5-min ultrahigh performance liquid chromatography-electrospray ionization mass spectrometry method (UHPLC-ESI MS) was developed. This method provided a wide linear range of quantification (4.8 nM to 60 µM for vicine, and 6.0 nM to 24 µM for convicine and levodopa), and low limit of detection (1.2 to 3.0 nM). The method was applied in the analysis of diverse faba bean samples, from dehulled mature faba bean and seed coat, to fresh plant tissues such as flowers, leaves, stems, roots and pods. The results indicated that fresh faba bean plant tissues, such as leaves and flowers, had the highest concentration of levodopa and low level of VC, which makes them an alternative source of plant-based l-DOPA with potential as a food-based treatment for Parkinson's Disease.

Green synthesis and characterization of silver nanoparticles from Ducrosia flabellifolia Boiss. aqueous extract: Anti-quorum sensing screening and antimicrobial activities against ESKAPE pathogens.

Biosynthesis of silver nanoparticles using natural compounds derived from plant kingdom is currently used as safe and low-cost technique for nanoparticles synthesis with important abilities to inhibit multidrug resistant microorganisms (MDR). ESKAPE pathogens, especially MDR ones, are widely spread in hospital and intensive care units. In the present study, AgNPs using Ducrosia flabellifolia aqueous extract were synthesized using a reduction method. The green synthesized D. flabellifolia-AgNPs were characterized by UV-Vis spectrophotometer, Scanning electron microscopy (SEM), and X-ray diffraction assays. The tested D. flabellifolia aqueous extract was tested for its chemical composition using Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS). Anti-quorum sensing and anti-ESKAPE potential of D. flabellifolia-AgNPs was also performed. Results from LC-ESI-MS technique revealed the identification of chlorogenic acid, protocatechuic acid, ferulic acid, caffeic acid, 2,5-dihydroxybenzoic acid, and gallic acid as main phytoconstituents. Indeed, the characterization of newly synthetized D. flabellifolia-AgNPs revealed spherical shape with mean particle size about 16.961±2.914 nm. Bio-reduction of silver was confirmed by the maximum surface plasmon resonance of D. flabellifolia-AgNPs at 430 nm. Newly synthetized D. flabellifolia-AgNPs were found to possess important anti-ESKAPE activity with low minimal inhibitory concentrations (MICs) ranging from 0.078 to 0.312 mg/mL, and low minimal bactericidal concentrations (MBCs) varying from 0.312 to 0.625 mg/mL. Moreover, D. flabellifolia-AgNPs were active against Candida utilis ATCC 9255, C. tropicalis ATCC 1362, and C. albicans ATCC 20402 with high mean diameter of growth inhibition at 5 mg/mL, low MICs, and minimal fungicidal concentrations values (MFCs). The newly synthetized D. flabellifolia-AgNPs were able to inhibit violacein production in Chromobacterium violaceum, pyocyanin in Pseudomonas aeruginosa starter strains. Hence, the newly synthesized silver nanoparticles using D. flabellifolia aqueous extract can be used as an effective alternative to combat ESKAPE microorganisms. These silver nanoparticles can attenuate virulence of Gram-negative bacteria by interfering with the quorum sensing system and inhibiting cell-to-cell communication.

Association of Blood Metabolomics Biomarkers with Brain Metabolites and Patient-Reported Outcomes as a New Approach in Individualized Diagnosis of Schizophrenia.

Given its polygenic nature, there is a need for a personalized approach to schizophrenia. The aim of the study was to select laboratory biomarkers from blood, brain imaging, and clinical assessment, with an emphasis on patients' self-report questionnaires. Metabolomics studies of serum samples from 51 patients and 45 healthy volunteers, based on the liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS), led to the identification of 3 biochemical indicators (cortisol, glutamate, lactate) of schizophrenia. These metabolites were sequentially correlated with laboratory tests results, imaging results, and clinical assessment outcomes, including patient self-report outcomes. The hierarchical cluster analysis on the principal components (HCPC) was performed to identify the most homogeneous clinical groups. Significant correlations were noted between blood lactates and 11 clinical and 10 neuroimaging parameters. The increase in lactate and cortisol were significantly associated with a decrease in immunological parameters, especially with the level of reactive lymphocytes. The strongest correlations with the level of blood lactate and cortisol were demonstrated by brain glutamate, N-acetylaspartate and the concentrations of glutamate and glutamine, creatine and phosphocreatine in the prefrontal cortex. Metabolomics studies and the search for associations with brain parameters and self-reported outcomes may provide new diagnostic evidence to specific schizophrenia phenotypes.

Identification of three unexpected new psychoactive substances at an Australian drug checking service.

Drug checking is a harm reduction measure that provides people with the opportunity to confirm the identity and purity of substances before consumption. The CanTEST Health and Drug Checking Service is Australia's first fixed-site drug checking service, where clients can learn about the contents of the samples they provide while receiving tailored harm reduction and health advice. Three samples were recently presented to the service with the expectation of 4-fluoromethylphenidate (4F-MPH) 1, methoxetamine (MXE) 2 and 3-methylmethcathinone (3-MMC) 3. The identity of all three samples did not meet these expectations and remained unknown on-site, as no high confidence identifications were obtained. However, further analysis by nuclear magnetic resonance spectroscopy, high resolution gas chromatography-electron ionisation-mass spectrometry and liquid chromatography-electrospray ionisation-mass spectrometry at the nearby Australian National University allowed for the structure elucidation of the three samples as 4-fluoro-α-pyrrolidinoisohexanophenone (4F-α-PiHP) 4, 1-(4-fluorobenzyl)-4-methylpiperazine (4F-MBZP) 5 and N-propyl-1,2-diphenylethylamine (propylphenidine) 6, respectively. Given all three samples were not of the expected identity and have not yet been described as new psychoactive substances in the literature, this study presents a full characterisation of each compound. As exemplified by this rapid identification of three unexpected new psychoactive substances, drug checking can be used as an effective method to monitor the unregulated drug market.

Isoflavone-Rich Extract of Trifolium resupinatum: Anti-obesity Attributes with In Silico Investigation of Its Constituents

Trifolium resupinatum L., Fabaceae, aqueous methanol leaf extract was selected to mitigate some obesity-associated risk factors to validate the possibility of further developing herbal drugs. Chromatography and spectrophotometric techniques verified 14 phenolics, five of which were first isolated from the plant and identified as 6''-O-acetyl ononin, genistin, daidzin, sissotrin, and astragalin. Further phytochemical characterization was performed via liquid chromatography-electrospray ionization-mass spectrometry assisted by a spectral similarity molecular network. In total, 81 metabolites were tentatively annotated including 69 species-first dereplications. Two major isolates (formononetin and pseudobaptigenin) were selected along with the investigated extract for an in vitro pancreatic lipase inhibition assay. They showed notable effects with IC50 values (µg/ml): 47.2 ± 1.1, 112.8 ± 1.23, and 471.32 ± 0.8, respectively, incomparable to orlistat (23.8 ± 0.64). Preliminary in vivo assay (25 mg/kg extract, daily, 8 weeks) displayed weight loss interest and promising advancement of serum triacylglycerides, total cholesterol, and glucose levels. Molecular docking studies confirmed the promising binding score of formononetin and pseudobaptigenin near the active sites and highlighted the affinity of other isolates to the lipase enzyme. Several isolates passed Lipinski’s law of the drug-likeness test, whereas SwissADME radar displayed that all constituents fall within the acceptable bioavailability zone. Therefore, the combination of flavonoids, especially isoflavones, could be regarded as drug-like agents for protection against obesity-induced metabolic complaints.Graphical

Streamlined Arsenolipid Identification via Direct Arsenic Detection Using RPLC-ESI-QTOF-MS with Collision-Induced Dissociation.

Arsenolipids are organoarsenicals with a long aliphatic chain that have been identified in a wide array of marine organisms. Precise analysis of arsenolipids is crucial for evaluating their toxicity, ensuring food safety, monitoring the environment, and gaining insights into the evolution of arsenic biogeochemistry. However, the discovery of new arsenolipids is often impeded by existing analytical challenges, notably the need for multiple instruments, such as the combination of liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) and inductively coupled plasma mass spectrometry (LC-ICP-MS). This study introduces a high-throughput untargeted analytical method on the basis of an unsophisticated instrumental configuration, LC-ESI-MS with collision-induced dissociation (CID) at 200 eV. This approach provides efficient dissociation of arsenic atoms from their precursor lipids and direct detection of the organic-bound arsenic as monatomic cations, As+. Application of this method has shown promise in rapidly characterizing arsenolipids in diverse samples, which has led to the discovery of a wide range of novel arsenolipids, including seven previously unidentified thioxoarsenolipids in ancient marine sediments.

Isolation, Antimicrobial Effect and Metabolite Analysis of Bacillus amyloliquefaciens ZJLMBA1908 against Citrus Canker Caused by Xanthomonas citri subsp. citri.

Citrus canker caused by Xanthomonas citri subsp. citri is a devastating bacterial disease with severe implications for the citrus industry. Microorganisms possessing biocontrol capabilities against X. citri subsp. citri offer a highly promising strategy for healthy citrus management. In the present study, a broad-spectrum antagonist strain ZJLMBA1908 with potent antibacterial activity against X. citri subsp. citri was isolated from symptomatic lemon leaves, and identified as Bacillus amyloliquefaciens. Cell-free supernatant (CFS) of strain ZJLMBA1908 also exhibited remarkable antimicrobial activity, especially suppressing the growth of X. citri subsp. citri and Nigrospora oryzae, with inhibition rates of 27.71% and 63.75%, respectively. The antibacterial crude extract (CE) derived from the CFS displayed effective activity against X. citri subsp. citri. A preventive treatment using the CE significantly reduced the severity and incidence of citrus canker in a highly susceptible citrus host. Additionally, the CE maintained activity in the presence of protease and under a wide range of temperature and pH treatments. Applying high-performance liquid chromatography (HPLC) to separate and purify the CE resulted in the discovery of one highly potent anti-X. citri subsp. citri subfraction, namely CE3, which could completely inhibit the growth of X. citri subsp. citri. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis revealed that CE3 mainly consisted of palmitic acid, surfactin C15, phytosphingosine and dihydrosphingosine. Taken together, the results contribute to the possible biocontrol mechanisms of B. amyloliquefaciens ZJLMBA1908, as well as providing a promising new candidate strain as a biological control agent for controlling citrus canker.

Proteome analysis of propolis deciphering the origin and function of its proteins

Propolis is a resinous honeybee product, rich in polyphenolic compounds and with high economic value. Although extensive studies regarding the chemical composition of different propolis extracts have been carried out, the propolis proteome remained unknown. The present study aimed at characterizing the proteome of two geographically-distinct propolis originating from Belgium and Iran. Propolis extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) followed by Matrix Assisted Laser Desorption Ionization – Time of Flight Mass Spectrometry (MALDI-TOF MS) and Liquid chromatography-electrospray ionization MS (LC-ESI-MS) analyses. Our comprehensive proteomic analysis led to the identification of honeybee venom and royal jelly proteins, and numerous plant-defense proteins in propolis, extending our understanding of the antimicrobial properties of this complex natural substance. The proteome of both propolis was mainly made up of proteins belonging to the poplar tree. Pathogenesis-related (PR) proteins, poplar PR-2 (β-1,3-glucanases) and PR-8 (acidic endochitinases) were found to be the major components of both propolis extracts, while the major bee venom allergen (Api m 1) and royal jelly proteins (MJRP 1, 2, 3 and 5) were identified in the propolis from Belgium. Overall, our proteome analyses revealed that propolis extracts from Belgium and Iran shared a core protein composition originating from the poplar proteome. The majority of identified proteins are involved in the plant defense against pathogens, most belonging to well-known pathogenesis-related families. Proteomics could thus represent a viable approach for origin authentication and allergen identification in poplar-type propolis.

Identification of nonvolatile organic compounds (NVOCs) in biopharmaceuticals through non-target analysis and quantification using complexation-precipitation extraction

Single-use systems in biopharmaceutical manufacturing can potentially release chemical constituents (leachables) into drug products. Prior to conducting toxicological risk assessments, it is crucial to establish the qualitative and quantitative methods for these leachables. In this study, we conducted a comprehensive screening and structure elucidation of 23 leachables (nonvolatile organic compounds, NVOCs) in two antibody drugs using multiple (self-built and public) databases and mass spectral simulation. We identified 7 compounds that have not been previously reported in medical or medicinal extractables and leachables. The confidence levels for identified compounds were classified based on analytical standards, literature references, and fragment assignments. Most of the identified leachables were found to be plasticizers, antioxidants, slip agents or polymer degradants. Polysorbate (namely Tween) is commonly used as an excipient for protein stabilization in biopharmaceutical formulations, but its ionization in liquid chromatography-electrospray ionization mass spectrometry can interfere with compound quantification. To address this, we employed a complexation-precipitation extraction method to reduce polysorbate content and quantify the analytes. The developed quantitative method for target NVOCs demonstrated high sensitivity (limit of quantification: 20 or 50 μg/L), accuracy (recoveries: 77.2 to 109.5 %) and precision (RSD ≤ 8.2 %). Overall, this established method will facilitate the evaluation of NVOC safety in drug products.

Redox-sensitive high-mobility group box-1 isoforms contribute to liver fibrosis progression and resolution in mice

High-mobility group box-1 (HMGB1) significantly increases and undergoes post-translational modifications (PTMs) in response to liver injury. Since oxidative stress plays a major role in liver fibrosis and induces PTMs in proteins, we hypothesized that redox-sensitive HMGB1 isoforms contribute to liver fibrosis progression and resolution. We used ESI-LC-MS (electrospray ionization-liquid chromatography-mass spectrometry) to study PTMs of HMGB1 during fibrosis progression and resolution. Conditional knockout mice were used for functional analyses. We identified that disulfide ([O]) and sulfonated ([SO3]) HMGB1 increase during carbon tetrachloride-induced liver fibrosis progression, however, while [O] HMGB1 declines, [SO3] HMGB1 drops but remains, during fibrosis resolution. Conditional knockout of Hmgb1 revealed that production of [O] and [SO3] HMGB1 occurs mostly in hepatocytes. Co-injection of [O] HMGB1 worsens carbon tetrachloride-induced liver fibrosis more than co-injection of [H] HMGB1. Conversely, ablation of [O] Hmgb1 in hepatocytes reduces liver fibrosis. Moreover, ablation of the receptor for advanced-glycation end-products (Rage) reveals that the profibrogenic effect of [O] HMGB1 is mediated by RAGE signaling in hepatic stellate cells (HSCs). Notably, injection of [SO3] HMGB1 accelerates fibrosis resolution due to RAGE-dependent stimulation of HSC apoptosis. Importantly, gene signatures activated by redox-sensitive HMGB1 isoforms in mice, classify patients with fibrosis according to fibrosis and inflammation scores. Dynamic changes in hepatocyte-derived [O] and [SO3] HMGB1 signal through RAGE-dependent mechanisms on HSCs to drive their profibrogenic phenotype and fate, contributing to progression and resolution of liver fibrosis. Since oxidative stress plays a major role in liver fibrosis and induces post-translational modifications of proteins, we hypothesized that redox-sensitive HMGB1 isoforms contribute to liver fibrosis progression and resolution. This study is significant because a rise in [H] HMGB1 could flag 'patient at risk', the presence of [O] HMGB1 could suggest 'disease in progress or active scarring', while the appearance of [SO3] HMGB1 could point at 'resolution under way'. The latter could be used as a readout for response to pharmacological intervention with anti-fibrotic agents.

Toxicological evaluation of ginsenoside-rich cell culture biomass of Panax japonicus produced in a large-scale bioreactor system

An environmentally friendly, resource-efficient substitute for the production of vegetative biomass and the bioactive components of medicinal plants is provided by novel biotechnology-based suspension cells cultured in bioreactors. Despite the cultured biomass is produced in the controlled conditions, it requires detailed chemical analysis and toxicological evaluation before being approved for human consumption. This study reports the results of biochemical, toxicological, and elemental composition analysis of the suspension cell culture of a medicinal species Panax japonicus (T. Nees) C.A. Mey. cultivated in a large-scale bioreactor system. For the scaling up the process of cultivation, the suspension culture was grown sequentially in a series of laboratory (20-L), pilot (75-L), and industrial (630-L) bioreactors. The biomass productivity of cell culture was comparable or higher than of wild plants or in vitro cultured hairy roots reported in the literature. The resulting cell biomass was analyzed for ginsenoside contents using ultra-high-performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC–ESI–MS). For the elemental composition of cells, inductively coupled argon plasma mass spectrometry (ICP–MS) was used. In addition, the biomass was evaluated for acute toxicity and skin sensibilization effects on in vivo animal models. The analyses confirmed that suspension cells cultured in bioreactors accumulate a broad spectrum of ginsenosides and their malonylated derivatives at a total concentration of 7.54 % (w/w) based on dry weight and contain essential macro- (K, Ca, Mg, Na) and micro- (Zn, Mn, Fe, B, Al, Cu) elements in dietary-safe concentrations. Acute toxicity tests using the administered doses of 2000 and 5000 mg dry biomass per kg animal weight resulted in no changes in animal organ weights and no or minor changes in hematological and biochemical parameters of blood. No skin irritation or sensitizing effects were observed. Therefore, bioreactor cultivation of P. japonicus cell suspension is highly productive and provides a ginsenoside-rich and non-toxic cell biomass which is a potentially valuable component of functional foods, food additives, and natural health products.

Condensed tannins from the leave of Dimocarpus longan Lour. exhibit anti-tyrosinase, anti-melanogenesis, anti-browning, anti-α-glucosidase, and anti-glycation activities

Dimocarpus longan Lour. is a valuable industrial crop for its broad applications in food, traditional Chinese medicine, and other fields. In this paper, condensed tannins were isolated from the leave of D. longan Lour., and their structure, anti-tyrosinase, anti-melanogenesis, anti-browning, anti-α-glucosidase, and anti-glycation properties were methodically clarified. The joint application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), electrospray ionization time-of-flight mass spectrometer (ESI+ TOF MS), and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) confirmed that the longan leave condensed tannins (LLCTs) were polymers of procyanidins (majority) and prodelphinidins. The polymers powerfully inhibit tyrosinase (TY) with a competitive mechanism. These compounds altered the microenvironment of the binding site through hydrophobic interaction and hydrogen bond, and caused conformational modification of the enzyme by changing the polypeptide framework and secondary structure, leading to a decrease in TY activity. Cell assays further suggested that LLCTs exhibited a strong inhibition against intracellular TY activity and melanin production, and caused apoptosis in B16F10 cells. Anti-browning studies demonstrated that LLCTs suppress the browning of fresh-cut apples. Furthermore, the LLCTs exhibited an impressive inhibition on both α-glucosidase and nonenzymatic glycation. Therefore, this study confirms the potential of LLCTs as new inhibitors of tyrosinase, melanogenesis, browning, α-glucosidase, and nonenzymatic glycation, which provided a theoretical basis for the potential utilization of these polymers in the cosmetics, pharmaceutical, and food industries.

The male's scent triggered a neural response in females despite ambiguous behavioral response in Asian house rats.

Pheromones and olfactory communication play vital roles in sex recognition and mate choice in rodents. Asian house rats (Rattus tanezumi) (RT) often startle easily, making behavioral measurements difficult to carry out accurately in the laboratory. Here, the behavioral and olfactory preferences of the female RT between males and females were not observed using a conventional two-choice device; we then explored the neuro-immunohistochemical evidence in the brains of RT females. We found that male urine elicited significantly higher c-fos expression in the accessory olfactory system and sex-related brain regions in females than female urine did. On the other hand, the differences of volatile compounds and major urinary proteins (MUPs) in both voided urine and preputial glands (PGs) of the RT were detected using gas chromatography-mass spectrometer, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and liquid chromatography-electrospray ionization mass spectrometry. We found that PG-derived 1-(4,5-dihydro-2-thiazolyl)-ethanone and total MUPs were more abundant in males versus females, suggesting these sexually divergent components might activate the female's accessory olfactory system. In conclusion, the neuro-immunohistochemical evidence indicated that potential sex pheromones might exist in RT; however, the strength of the chemical signal might be too weak to cause behavioral responses in females.

Preparation of low molecular weight chondroitin sulfate from different sources by H2O2/ascorbic acid degradation and its degradation mechanism

Low molecular weight chondroitin sulfate (LMCS) has attention for enhanced bioavailability and bioactivity compared to native CS. We optimized H2O2/ ascorbic acid (Vc) degradation conditions to prepare LMCS from chicken, bovine, and shark cartilages. Degradation kinetics models and chemical composition data of LMCS showed the GlcA residues of chondroitin-4-sulfate (CSA) may be preferentially attacked. Nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography-electrospray mass spectrometry (HPLC-MS) indicated that the CH of GlcA in CS was broken through a hydrogen abstraction reaction to break the β-(1 → 3) bond and form the hexendioic acid product. Standard density functional theory (DFT) calculations indicated that the energy required for the hydrogen abstraction from the C1-H bond in GlcA was lower than that of GalNAc. Molecular dynamics (MD) showed that CSA was more likely to interact with hydroxyl radicals (·OH) than non-sulfated chondroitin (CSO) and chondroitin-6-sulfate (CSC). These results provide guidance for producing LMCS.

Profiling of regioisomeric triacylglycerols in pistachio nuts by high-performance liquid chromatography-electrospray ionization mass spectrometry

In this contribution, a non-aqueous reversed-phase liquid chromatographic method coupled to electrospray ionization (ESI) tandem mass spectrometry (LC-MS) is used for the identification of triacylglycerol (TAG) down to the regioisomersíc level in pistachio lipids. Furthermore, for effective profiling of TAG, a two-dimensional (2D) map was employed to identify the entire homologous series of TAGs especially, odd-numbered fatty acid and long-chain derivatives. Notably, TAGs were observed as pairs of regioisomers with distinct retention times and tandem mass spectra. Furthermore, more than 100 TAGs containing 26 fatty acids with 51-63 carbon atoms and 0-3 double bonds were identified in different pistachio samples. The developed method is effective and facile for the fast quantification of complex TAGs in pistachio samples at high selectivity.