Trace determination and characterization of ginsenosides in rat plasma through magnetic dispersive solid-phase extraction based on core-shell polydopamine-coated magnetic nanoparticles

Enrichment of trace bioactive constituents and metabolites from complex biological samples is challenging.This study presented a one-pot synthesis of magnetic polydopamine nanoparticles(Fe3O4@-SiO2@PDA NPs)with multiple recognition sites for the magnetic dispersive solid-phase extraction(MDSPE)of ginsenosides from rat plasma treated with white ginseng.The extracted ginsenosides were characterized by combining an ultra-high-performance liquid chromatography coupled to a highresolution mass spectrometry with supplemental UNIFI libraries.Response surface methodology was statistically used to optimize the extraction procedure of the ginsenosides.The reusability of Fe3O4@-SiO2@PDA NPs was also examined and the results showed that the recovery rate exceeded 80%after recycling 6 times.Furthermore,the proposed method showed greater enrichment efficiency and could rapidly determine and characterize 23 ginsenoside prototypes and metabolites from plasma.In comparison,conventional methanol method can only detect 8 ginsenosides from the same plasma samples.The proposed approach can provide methodological reference for the trace determination and characterization of different bioactive ingredients and metabolites of traditional Chinese medicines and food.

Therapeutic Effects of Selaginella tamariscina on the Model of Acute Gout with Hyperuricemia in Rats Based on Metabolomics Analysis

Gout is a disease of purine metabolic disorders which results from long-term hyperuricemia and the sodium urate deposition in and around the joints. Selaginella tamariscina （ST） is an important traditional Chinese herbal medicine and is used for the treatment of gout and hyperuricemia. In this study, the rat model of acute gout with hyperuricemia was established by intraperitoneal injection of xanthine and oxonic acid potassium salt and articular injection monosodium urate （MSU）. The effect of ST in the treatment of gout was investigated by measuring joint swelling, the expression of IL-1 !3 in serum and histological changes of joint by haematoxylin eosin （H＆E） staining. Subsequently, urine metabolomics analysis for biomarkers discovery in acute gout with hyperuricemia rats was performed by the ultra-performance liquid chromatography-electrospray ionization quadruple time-of-flight mass spectrometry （UPLC-ESI-QTOF/MS） combined with chemometric approach. Principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA） were used to detect potential biomarkers. A total of 18 potential biomarkers were identified mainly including tryptophan metabolism; tyrosine metabolism; ly- sine methylation; pyrimidine metabolism; purine metabolism; TCA cycle and fatty acid metabolisms. This study in- dicates that ST could efficiently ameliorate the disease of acute gout with hyperuricemia in rats. The related meta- bolic biomarkers could provide useful information and the metabolic mechanism could be used for further study about the model of acute gout with hyperuricemia in rats.

Mechanism of Incompatible Herb Pairs, Panax ginseng and Veratrum nigrum L,： Material Basis and Metabolic Profiles of Ginsenosides in Rat Intestinal Bacteria

In traditional Chinese medicine theory, Panax ginseng and Veratrum nigrum L. is an important incompatible herb pair. Studies on the content variation of main components and the influences on the metabolism in rat intestinal bacteria are useful to understand the mechanism of incompatibility of this herb pairs. In this study, the content variation of ginsenosides and their metaboltic profiles in the extracts of P. ginseng and compatibility of P. ginseng with V. nigrum L. （G-V） were investigated using relative quantitative method of electrospray ionization mass spec- trometry （ESI-MS） and UPLC-MSn, respectively. The relative contents of most ginsenosides were reduced in the extract of G-V. Furthermore, ginsenosides Rbt, Rb2, Rc and Rd could be metabolized to Rd, F2 and C-K in rat in- testinal bacteria. The metabolic speeds ofRbl, Rb2 and Re in the G-V extracts at ratios of 10 ： 5, 10 ： 7 and 10 ： 10 and the metabolic rates of ginsenosides Rbb Rb2 and Rc to Rd, Rd to F2 in all compatibility extracts were lower than that in the P ginseng extract. In conclusion, this study illustrated the mechanism of effect-reducing by comparison of the relative contents and metabolic profiles of ginsenosides after compatibility of P ginseng and V. nigrum L.

α-Glucosidase immobilization on functionalized Fe_3O_4 magnetic nanoparticles for screening of enzyme inhibitors

Magnetic nanoparticles(MNPs) are widely used for the immobilization of enzyme owing to the unique properties such as good biocompatibility and rapid separation. Herein, we used Fe_3O_4 magnetic nanoparticles(Fe_3O_4 MNPs) as the carrier core with(3-aminopropyl)triethoxysilane(APTES)modification by our approach, in which a-glucosidase was stereoscopically immobilized on the surface of Fe_3O_4 MNPs via covalent binding. The result of immobilization was characterized by scanning electron microscope(SEM) and fourier transform-infrared spectroscopy(FT-IR). Then we optimized some key parameters of the immobilization reaction, including the ratio of MNPs to enzyme, GA concentration,crosslinking time and immobilization time. Moreover, under the optimal conditions, pH tolerance,thermo stability and reusability of the immobilized enzyme were investigated and compared with the free one. In order to evaluate the change of the affinity of the enzyme to its specific substrate after immobilization, the Michaelis-Menten constant(K_m) was also studied. Finally, the immobilized α-glucosidase combining with high performance liquid chromatography-tandem mass spectrometry technique(HPLC-MS/MS) was applied to screen and identify eight inhibitors from Polygonum cuspidatum extract. These results indicated that the established method had the broad prospects for biotechnological applications.

In Situ Analysis for Herbal Pieces of Aconitum Plants by Using Direct Analysis in Real Time Mass Spectrometry

In this study,an extend application was developed to in situ analyze the herbal pieces of Aconitum plants by Direct Analysis in Real Time Mass Spectrometry(DART-MS).Nearly all aconitine-type alkaloids can be desorbed and ionized in this method,including diester diterpenoid aconitines(DDAs),monoester diterpenoid aconitines(MDAs)and some other diterpenoid aconitines.The spectra of in situ analysis for the herbal pieces of aconitum plants are similar with that of their extracts.Radix Aconiti and Radix Aconiti Kusnezoffii can be distinguished from each other by the intensity differences of character fragment ions from MDAs,such as m/z 586,572,and 556.The qualified and unqualified herbal pieces can be also identified by the relative abundances of DDAs.The RSD of the relative abundances of some character ions at m/z 556,586,and 590 were 13.53%,4.03%,and 12.03%,respectively.So this in situ analytical method can identify both the types of Aconitum preparata and their quality.It provides the following advantages in the analysis of Chinese herbs:fast detection without much pretreatment,high-throughput analysis,and reduction of pollution without any organic solvent.

Analysis and Identification of the Chemical Constituents of Ding-Zhi-Xiao-Wan Prescription by HPLC-IT-MSn and HPLC-Q-TOF-MS

Ding-Zhi-Xiao-Wan(DZXW)is a famous traditional Chinese medicine(TCM)formula,which is composed of four herbs,Ginseng Radix,Poria,Polygala Radix and Acori Tatarinowii Rhizoma.It has been popularly used for the treatment of emotional disease,like Alzheimer’s disease,Parkinson’s disease,depression,anxiety,forgetfulness and neurasthenia.In this research,a high-performance liquid chromatography coupled with ion-trap tandem mass spectrometry(HPLC-IT-MS^(n))method along with a high-performance liquid chromatography coupled with quadru-pole time-of-flight mass spectrometry(HPLC-Q-TOF-MS)method in negative ion mode was established to inves-tigate the major constitutions in DZXW.The extracts were prepared by ultra-sonication in ethyl acetate,n-butanol,95%ethanol and deionized water sequentially as well as in deionized water directly.A Kromasil C18 column was used to separate the extracts of DZXW.Acetonitrile and 0.1%aqueous formic acid(V/V)were used as the mobile phase.A total of 64 components were characterized,including 16 triterpenoids,14 Polygala saponins,10 oligosac-charide esters,6 sucrose esters,2 xanthone C-glycosides and 16 ginsenosides.

Effects of the ambient fine particulate matter(PM2.5) exposure on urinary metabolic profiles in rats using UPLC-Q-TOF-MS

In order to study the damage of ambient fine particulate matter(PM2.5) to organism in the environment,the method of urinary metabolomics was used to detect the potential endogenous metabolites and their related metabolic pathways to clarify the toxicity mechanisms. Particles of PM2.5 collected from the fibrous membrane were extracted ultrasonically and dissolved in physiological saline to prepare PM2.5 suspension. The solution of saline and fine particular matter was injected into the trachea of rats respectively. The rats were injected two times a week for four weeks. Samples of 24-h urine were collected at the 14 th day after the end of exposure, and the ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used for the metabolomics detection. Principal component analysis(PCA) was used to investigate the global metabolomic alterations and a clear separation in the scatter diagram were observed. 17 potential endogenous metablites were identified from urinary samples in rats by UPLC-Q-TOF-MS. The primary metabolism pathways involved pentose and glucuronate interconversions, starch and sucrose metabolism, tryptophan metabolism, tyrosine metabolism, phenylalanine metabolism, purine metabolism, acetaminophen metabolism pathway, retinol metabolism and valproic acid metabolism pathway.The results are helpful to understand the toxicological mechanisms of PM2.5 and screened out potential biomarkers of rats which exposure to PM2.5.

A novel strategy for high-specificity, high-sensitivity, and high-throughput study for gut microbiome metabolism of aromatic carboxylic acids

Aromatic carboxylic acids(ACAs) may be as transformed key metabolites via gut microbiome for playing better pharmacological effects. However, it's rare to achieve high-specificity, high-sensitivity, and highthroughput detection simultaneously, especially, for tracing trace ACAs in gut microbiome. In this work,firstly, a novel dual-template and double-shelled molecularly imprinted 96-well microplates(DDMIPs)was designed and amplified signal for p-hydroxybenzoic acid(PBA) and 3,4,5-trimethoxycinnamic acid(TMA). Additionally, the DDMIPs and a stable isotope labeling derivatization(SILD) method combined with the ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry(UHPLC-TQ MS) was firstly stepwise integrated, achieving high-effective, high-sensitive, and highthroughput study of gut microbiome metabolism. The whole strategy showed lower limits of detections(LODs) up to 1000 folds than the traditional method, and revealed a more real metabolism-time profile of PBA and TMA by 3-step signal amplification. The platform also laid the foundation for fast, simple,high-selective, high-effective, and high-throughput metabolism and pharmacological research.

Separation, Quantification and Structural Study of (+)-Catechin and (-)-Epicatechin by Ion Mobility Mass Spectrometry Combined with Theoretical Algorithms

Two catechin epimers and their non-covalent complexes with γ-cyclodextrin were studied by using ion mobility coupled with mass spectrometry (IM-MS). Rapid separation of complexes was achieved with the peak-to-peak resolution reaching 0.86 after optimization of IM condition. Collision cross section (CCS) was measured to explore the structural difference of complexes. A gap of 11.75 A^2 between two complexes was found. Molecular modeling and theoretical CCS calculation were adopted to explain the measurement results. Two binding ways of both complexes were found and the calculated CCS corresponds accurately to the measured CCS. Quantification of catechins in mixtures was performed and the relative error was less than 15%, indicating the effectiveness of quantification by IM-MS.