液相色谱/光谱(紫外、质谱及核磁共振)联用技术在中草药有效成分研究中的应用

本文综述了液相色谱／光谱(紫外、质谱及核磁共振)联用技术的最新进展及其在中草药有效成分研究中的应用。并对这一新技术在实际应用中的优缺点及应用前景进行了分析和展望。     

A strategy for modelling dynamic responses in metabolic samples characterized by GC/MS

A multivariate strategy for studying the metabolic response over time in urinary GC/MS data is presented and exemplified by a study of drug-induced liver toxicity in the rat. The strategy includes the generation of representative data through hierarchical multivariate curve resolution (H-MCR), highlighting the importance of obtaining resolved metabolite profiles for quantification and identification of exogenous (drug related) and endogenous compounds (potential biomarkers) and for allowing reliable comparisons of multiple samples through multivariate projections. Batch modelling was used to monitor and characterize the normal (control) metabolic variation over time as well as to map the dynamic response of the drug treated animals in relation to the control. In this way treatment related metabolic responses over time could be detected and classified as being drug related or being potential biomarkers. In summary the proposed strategy uses the relatively high sensitivity and reproducibility of GC/MS in combination with efficient multivariate curve resolution and data analysis to discover individual markers of drug metabolism and drug toxicity. The presented results imply that the strategy can be of great value in drug toxicity studies for classifying metabolic markers in relation to their dynamic responses as well as for biomarker identification.     

Probiotic modulation of symbiotic gut microbial–host metabolic interactions in a humanized microbiome mouse model

The transgenomic metabolic effects of exposure to either Lactobacillus paracasei or Lactobacillus rhamnosus probiotics have been measured and mapped in humanized extended genome mice (germ‐free mice colonized with human baby flora). Statistical analysis of the compartmental fluctuations in diverse metabolic compartments, including biofluids, tissue and cecal short‐chain fatty acids (SCFAs) in relation to microbial population modulation generated a novel top‐down systems biology view of the host response to probiotic intervention. Probiotic exposure exerted microbiome modification and resulted in altered hepatic lipid metabolism coupled with lowered plasma lipoprotein levels and apparent stimulated glycolysis. Probiotic treatments also altered a diverse range of pathways outcomes, including amino‐acid metabolism, methylamines and SCFAs. The novel application of hierarchical‐principal component analysis allowed visualization of multicompartmental transgenomic metabolic interactions that could also be resolved at the compartment and pathway level. These integrated system investigations demonstrate the potential of metabolic profiling as a top‐down systems biology driver for investigating the mechanistic basis of probiotic action and the therapeutic surveillance of the gut microbial activity related to dietary supplementation of probiotics.     

An innovative LC/MS approach applied to the determination of zeralenone in maize: Alternate Isotope-coded Derivatization Assay (AIDA)

Zearalenone is a mycotoxin mainly produced by severalFusarium species, which are known to colonize grains in temperate climates. The purpose of the study is to provide a reliable isotope dilution method for the quantification of this mycotoxin. A derivative of the analyte to be used as standard is obtained by reaction with acetic anhydride, which is available in two pure isotopic forms, a protonated (“light”) and a hexadeuterated (“heavy”). The derivatized standards are added to the matrix split intwo parts. Then, the derivatization procedure is repeated on both matrices derivatizing the part containing the “heavy” labelled standard with the “light” acetic anhydride and the part containing the “light” labelled standard with the “heavy” acetic anhydride. Both extracted mixtures are analyzed by LC/MS, monitoring the “light” and the “heavy” labelled analytes and using the former as standard for the latter in one case and viceversa in the other case. The method allowed to obtain very good results, without the need of IAC purification. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005. Financial support: The Italian Ministry of Health     

Phytochemical Fingerprints and Bioactivities of Ripe Disseminules (Fruit-Seeds) of Seventeen Gundelia (Kenger-Kereng Dikeni) Species from Anatolia with Chemometric Approach

Gundelia species are known as “Kenger-kereng dikeni” in Anatolia, and their aerial parts are consumed as food. Also, roots and seeds (disseminules) of the Gundelia species are used to prepare gum and coffee. The chemical contents of ethanol and hexane extracts of disseminules of 17 Gundelia species, 13 of them are endemic, were studied using LC/MS/MS and GC/MS. Additionally, their antioxidant potential and enzyme inhibitory capacity against acetyl- and butyryl-cholinesterase, urease, and tyrosinase were determined. The unsaturated fatty acid ratios of Gundelia species were higher than their saturated fatty acid ratio. The highest sum of oleic and linoleic acid was detected in G. tournefortii var. tenuisecta (70.42 %). β-Sitosterol, α-amyrin, 3-acetyllupeol were identified in 17 Gundelia species by GC/MS, while chlorogenic acid and luteolin by LC/MS/MS as major compounds. The ethanol and hexane extracts of G. siirtica, G. rosea, and G. mesopotamica indicated good cholinesterase inhibitory activity. Among all species, ethanol extract of G. colemerikensis exhibited the best activity in ABTS (IC₅₀: 32.30±0.98 μg/mL), DPPH (IC₅₀: 59.91±0.89 μg/mL), and CUPRAC (A_0.5: 57.41±1.03 μg/mL) assays. Ethanol extract of G. colemerikensis also displayed the highest inhibitory activity against butyrylcholinesterase (51.14±0.25 % at 200 μg/mL), urease (51.71±1.75 % at 200 μg/mL), and tyrosinase (39.50±0.85 % at 200 μg/mL) enzymes. According to the chemometric analysis of fatty acids, four groups were observed. Therefore, it is suggested that G. colemerikensis can be used in the pharmaceutical, food, and cosmetic industries due to its antioxidant and enzyme inhibition properties.     

Metabolic engineering of flavonoids in tomato (Solanum lycopersicum): the potential for metabolomics

Flavonoids comprise a large and diverse group of polyphenolic plant secondary metabolites. In plants, flavonoids play important roles in many biological processes such as pigmentation of flowers, fruits and vegetables, plant-pathogen interactions, fertility and protection against UV light. Being natural plant compounds, flavonoids are an integral part of the human diet and there is increasing evidence that dietary polyphenols are likely candidates for the observed beneficial effects of a diet rich in fruits and vegetables on the prevention of several chronic diseases. Within the plant kingdom, and even within a single plant species, there is a large variation in the levels and composition of flavonoids. This variation is often due to specific mutations in flavonoid-related genes leading to quantitative and qualitative differences in metabolic profiles. The use of such specific flavonoid mutants with easily scorable, visible phenotypes has led to the isolation and characterisation of many structural and regulatory genes involved in the flavonoid biosynthetic pathway from different plant species. These genes have been used to engineer the flavonoid biosynthetic pathway in both model and crop plant species, not only from a fundamental perspective, but also in order to alter important agronomic traits, such as flower and fruit colour, resistance, nutritional value. This review describes the advances made in engineering the flavonoid pathway in tomato (Solanum lycopersicum). Three different approaches will be described; (I) Increasing endogenous tomato flavonoids using structural or regulatory genes; (II) Blocking specific steps in the flavonoid pathway by RNA interference strategies; and (III) Production of novel tomato flavonoids by introducing novel branches of the flavonoid pathway. Metabolite profiling is an essential tool to analyse the effects of pathway engineering approaches, not only to analyse the effect on the flavonoid composition itself, but also on other related or unrelated metabolic pathways. Metabolomics will therefore play an increasingly important role in revealing a more complete picture of metabolic perturbation and will provide additional novel insights into the effect of the introduced genes and the role of flavonoids in plant physiology and development.     

Identification of antioxidant phenolic compounds in feverfew (Tanacetum parthenium) by HPLC-ESI-MS/MS and NMR

Antioxidant polyphenolic acids in the medicinal herb feverfew (Tanacetum parthenium) were isolated through in vitro bioassay-orientated antioxidant tests in response to 1,1-diphenyl-2-picrylhydrazyl (DPPH*) free radical scavenging and Fe(2+)-chelating activities. Purification of the active compounds and their structural elucidation involved a variety of techniques including open-column chromatography, HPLC, GC-MS, LC-MS and NMR. Major compounds with potent DPPH* scavenging activities were characterised as 3,5-, 4,5- and 3,4-di-O-caffeoylquinic acids (DCQAs). This is the first report of DCQAs found in feverfew.     

SATE (Aryl) Phosphotriester Series. II. Stability Studies and Physicochemical Parameters

Abstract

The stability of phosphotriester derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and various aryl residues derived from L-tyrosine was evaluated in biological media. The results demonstrate that such compounds give rise to intracellular delivery of the parent mononucleotide through esterase and phosphodiesterase hydrolytic steps, successively.     

Identification of the major glucosinolate (4-mercaptobutyl glucosinolate) in leaves of Eruca sativa L. (salad rocket)

The major and structurally unique glucosinolate (GLS) in leaves of Eruca sativa L. (salad rocket) was identified as 4-mercaptobutyl GLS. Both 4-methylthiobutyl GLS and 4-methylsulfinylbutyl GLS were also present, but at lower concentrations. The 4-mercaptobutyl GLS was observed to oxidise under common GLS extraction conditions, generating a disulfide GLS that may be reduced efficiently by tris(2-carboxyethyl) phosphine hydrochloride (TCEP) to reform the parent molecule. The identities of 4-mercaptobutyl GLS and of the corresponding dimeric GLS were confirmed by LC/MS, MS/MS and NMR. Myrosinase treatment of an enriched GLS fraction or of the purified dimer GLS generated a mixture of unique bi-functional disulfides, including bis-(4-isothiocyanatobutyl) disulfide (previously identified elsewhere). TCEP reduction of the purified dimer, followed by myrosinase treatment, yielded only 4-mercaptobutyl ITC. GLS-derived volatiles generated by autolysis of fresh seedlings and true leaves were 4-mercaptobutyl ITC (from the newly identified GLS), 4-methylthiobutyl ITC (from 4-methylthiobutyl GLS) and 4-methylsulfinylbutyl ITC (from 4-methylsulfinyl-butyl GLS); no unusual bi-functional disulfides were found in fresh leaf autolysate. These results led to the conclusion that, in planta, the new GLS must be present as 4-mercaptobutyl GLS and not as the disulfide found after extraction and sample concentration. This new GLS and its isothiocyanate are likely to contribute to the unique odour and flavour of E. sativa.     

The Sperm Proteome of the Echiuran Urechis unicinctus (Annelida,Echiura)

Sperm proteins presumably play critical roles in reproduction, but in many non‐model animals their identities are unknown. A total of 147 sperm proteins from the echiuran worm Urechis unicinctus, the first sperm proteome in the phylum Annelida, are reported. The echiuran sperm proteome can be classified into diverse functional groups: energy metabolism (31%), protein synthesis and degradation (18%), spermatogenesis and sperm motility (12%), signal pathway (11%), ion channel and transport proteins (6%), cytoskeleton (4%), immunity and stress responses (3%), and fertilization (1%). These results will facilitate studies of mechanisms of fertilization in echiurans, as well as comparative studies of reproduction and evolution across lophotrochozoans. Data are available via ProteomeXchange with identifier PXD009176.     

Determination of oxcarbazepine and its metabolites in postmortem blood and hair by means of liquid chromatography with mass detection (HPLC/APCI/MS)

A typical use of hair analysis in forensic toxicology is the documentation of previous drug administration. This is illustrated in a suicidal death of a 58-year-old epileptic patient who was treated with oxcarbazepine and probably with levomepromazine. The toxicological analysis carried out by HPLC/APCI/MS included also the hair (6 cm length) besides postmortem blood. The method was validated for levomepromazine, oxcarbazepine (OXCBZ) and its two metabolites, 10-hydroxycarbazepine (CBZ-10OH) and trans-diol-carbazepine (CBZ-diOH) in various biological matrices. The analysis of the postmortem blood indicated oxcarbazepine and its two main metabolites were present at therapeutic concentrations; levomepromazine was detected at a fatal concentration. In three 2-cm segments of hair, oxcarbazepine and its two metabolites were detected; however, levomepromazine was not detected in this specimen. As a result of complex chemical-toxicological investigation it was confirmed the information that the decedent. was an epileptic patient and was treated with oxcarbazepine for at least 6 months before death. In addition, he took a toxic dose of levomepromazine in order to commit suicide. The analysis revealed differences between the concentration levels of oxcarbazepine and its active metabolite CBZ-10OH in postmortem specimens and hair, suggesting different mechanisms of penetration of metabolites and their precursors into this matrix.     

Differential metabolic response of cultured rice (Oryza sativa) cells exposed to high‐ and low‐temperature stress

Global mean temperatures are expected to rise by 2–4.5°C by 2100, accompanied by an increase in frequency and amplitude of extreme temperature events. Greater climatic extremes and an expanded range of cultivation will expose rice to increasing stress in the future. Understanding gene expression in disparate thermal regimes is important for the engineering of cultivars with tolerance to nonoptimal temperatures. Our study investigated the proteomic responses of rice cell suspension cultures to sudden temperature changes. Cell cultures grown at 28°C were subjected to 3‐day exposure to 12 or 20°C for low‐temperature stress, and 36 or 44°C for high‐temperature stress. Quantitative label‐free shotgun proteomic analysis was performed on biological triplicates of each treatment. Over 1900 proteins were expressed in one or more temperature treatments, and, of these, more than 850 were found to be responsive to either of the temperature extremes. These temperature‐responsive proteins included more than 300 proteins which were uniquely expressed at either 12 or 44°C. Our study also identified 40 novel stress–response proteins and observed that switching between the classical and the alternative pathways of sucrose metabolism occurs in response to extremes of temperature.     

Integration of LC/MS,NMR and Molecular Docking for Profiling of Bioactive Diterpenes from Euphorbia mauritanica L. with in Vitro Anti-SARS-CoV-2 Activity

In spite of tremendous efforts exerted in the management of COVID-19, the absence of specific treatments and the prevalence of delayed and long-term complications termed post-COVID syndrome still urged all concerned researchers to develop a potent inhibitor of SARS-Cov-2. The hydromethanolic extracts of different parts of E. mauritanica were in vitro screened for anti-SARS-Cov-2 activity. Then, using an integrated strategy of LC/MS/MS, molecular networking and NMR, the chemical profile of the active extract was determined. To determine the optimum target for these compounds, docking experiments of the active extract's identified compounds were conducted at several viral targets. The leaves extract showed the best inhibitory effect with IC₅₀ 8.231±0.04 μg/ml. The jatrophane diterpenes were provisionally annotated as the primary metabolites of the bioactive leaves extract based on multiplex of LC/MS/MS, molecular network, and NMR. In silico studies revealed the potentiality of the compounds in the most active extract to 3CLpro, where compound 20 showed the best binding affinity. Further attention should be paid to the isolation of various jatrophane diterpenes from Euphorbia and evaluating their effects on SARS-Cov-2 and its molecular targets.     

Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia)

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick spore wall composed of a glycoprotein-rich outer layer or exospore and a chitin-rich inner layer or endospore. In this study performed on the silkworm pathogen Nosema bombycis, we analyzed the spore wall proteins (SWPs) by proteomic-based approaches, MALDI-TOF MS and LC-MS/MS, and 14 hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. Furthermore, we have examined the SWPs by SDS-PAGE and three main spore wall peptides were detected with molecular weights of 32.7 kDa (SWP32), 30.4 kDa (SWP30), and 25.3 kDa (SWP25), respectively. By N-terminal amino acid residue sequencing, and searching the genomic DNA shotgun database of N. bombycis, the complete ORFs of SWP30 and SWP32 were obtained, which encode for a 278- and a 316-amino acid peptide, respectively. Mouse polyclonal antibodies were raised against SWP30 and SWP32 recombinant proteins produced in Escherichia coli, and the results of indirect immunofluorescence assay (IFA) and immunoelectron microscopy (IEM) analyses indicated SWP30 to be an endosporal protein while SWP32 was shown to be an exosporal protein. Both SWP30 and SWP32 are included in the 14 HSWPs identified by MS, confirming the results of the proteomic-based approaches.     

Glutathionylated 4-hydroxy-2-(E)-alkenal enantiomers in rat organs and their contributions toward the disposal of 4-hydroxy-2-(E)-nonenal in rat liver

The major route for elimination of 4-hydroxy-2-(E)-nonenal (4-HNE) has long been considered to be through glutathionylation and eventual excretion as a mercapturic acid conjugate. To better quantitate the glutathionylation process, we developed a sensitive LC–MS/MS method for the detection of glutathione (GSH) conjugates of 4-hydroxy-2-(E)-alkenal enantiomers having a carbon skeleton of C5 to C12. The newly developed method enabled us to quantify 4-hydroxy-2-(E)-alkenal–glutathione diastereomers in various organs, i.e., liver, heart, and brain. We identified the addition of iodoacetic acid as a critical step during sample preparation to avoid an overestimation of glutathione–alkenal conjugation. Specifically, we found that in the absence of a quenching step reduced GSH and 4-hydroxy-2-(E)-alkenals react very rapidly during the extraction and concentration steps of sample preparation. Rat liver perfused with d₁₁-4-hydroxy-2-(E)-nonenal (d₁₁-4-HNE) revealed enantioselective conjugation with GSH and transportation out of the liver. In the d₁₁-4-HNE-perfused rat livers, the amount of d₁₁-(S)-4-HNE–GSH released from the rat liver was higher than that of d₁₁-(R)-4-HNE–GSH, and more d₁₁-(R)-4-HNE–GSH than d₁₁-(S)-4-HNE–GSH remained in the perfused liver tissues. Overall, the glutathionylation pathway was found to account for only 8.7% of the disposition of 4-HNE, whereas catabolism to acetyl-CoA, propionyl-CoA, and formate represented the major detoxification pathway.     

Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

We have studied rapid and simple sugar mapping using liquid chromatography/electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column. The oligosaccharide mixture was separated on the basis of the sequence, branching structure, and linkage, and each oligosaccharide was characterized based on its molecular mass. In this study we demonstrated the usefulness of capillary LC/MS (CapLC/MS) and capillary liquid chromatography/tandem mass spectrometry (CapLC/MS/MS) as sensitive means for accomplishing the structural analysis of oligosaccharides in a low-abundance glycoprotein. The carbohydrate heterogeneity and molecular mass information of each oligosaccharide can be readily obtained from CapLC/MS of a small amount of glycoprotein. CapLC/MS/MS provided b-ion series, which is informative with regard to monosaccharide sequence. Exoglycosidase digestion followed by CapLC/MS elucidated a carbohydrate residue linkage. Using this method, we characterized N-linked oligosaccharides in hepatocyte growth factor produced in mouse myeloma NS0 cells as the complex-type bi-, tri-, and tetraantennary terminated with N-glycolylneuraminic acids and alpha-linked galactose residues. Sugar mapping with CapLC/MS and CapLC/MS/MS is useful for monitoring glycosylation patterns and for structural analysis of carbohydrates in a low-abundance glycoprotein and thus will become a powerful tool in biological, pharmaceutical, and clinical studies.     

Isolation of polyketides from Prymnesium parvum (Haptophyta) and their detection by liquid chromatography/mass spectrometry metabolic fingerprint analysis

Prymnesium parvum is a microalga that forms blooms coupled with the presence of potent exotoxins; however, no chemical standards are currently available for the toxins. Streamlined methods are presented for the separation and enrichment of polyketide toxins, prymnesin-1 (prym1) and prymnesin-2 (prym2). Prymnesins were separated by reversed-phase chromatography and detected by positive-mode electrospray ionization MS to generate a unique metabolic fingerprint. More than 10 ions were detected and mass assignments were in agreement with predicted isotopic distributions for the intact compounds and related fragments; ions occurred as multiply protonated species and with common salt adducts. The most prevalent ion was observed at 919.88 m/z, which represents the aglycone [prym_agly + 2H]²⁺ backbone structure common to both molecules. Expanded mass spectra for this and related ions were in excellent agreement (<0.5 ppm) with empirically derived spectra based on elemental composition and naturally occurring isotopes. These investigations have confirmed the isolation of polyketide prymnesins from whole cells, which heretofore has not been reproduced since their original characterization. Moreover, this study represents the first time these compounds have been verified in aqueous materials. These tools should allow the direct identification and analysis of polyketide prymnesins, which will greatly improve our understanding of these toxins in P. parvum.     

Validation and implementation of a method for determination of bryostatin 1 in human plasma by using liquid chromatography/tandem mass spectrometry

A new sensitive and specific method using liquid chromatography/tandem mass spectrometry for determination of bryostatin 1 was developed and validated. Sample pretreatment involved a double liquid-liquid extraction step with a mixture of acetonitrile/n-butyl chloride (1/4, v/v). Separation of the compound of interest, including the internal standard paclitaxel, was achieved on a Waters X-Terra C18 (50 x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (80:20, v/v) containing 0.1% formic acid using isocratic flow at 0.15 mL/min for 13 min. The analytes of interest were monitored by tandem mass spectrometry with electrospray positive ionization. The linear calibration curves were generated over the range of 50-2000 pg/mL with values for the coefficient of determination of >0.99. The values for both within-day and between-day precision and accuracy were <15%. This method was used to characterize the plasma pharmacokinetics of bryostatin 1 at doses of 20 microg/m2) to optimize treatment with this agent.