Cultivar differentiation of Citri Reticulatae Pericarpium by a combination of hierarchical three-step filtering metabolomics analysis, DNA barcoding and electronic nose.

The traditional Chinese medicine Citri Reticulatae Pericarpium (CRP) was mainly originated from the dried pericarp of Citrus reticulata 'Chachi' (Crc), Citrus reticulata 'Dahongpao' (Crd), Citrus reticulata 'Unshiu' (Cru) and Citrus reticulata 'Tangerina' (Crt) in China. Since these four cultivars have great similarities in morphology, reliable methods to differentiate CRP cultivars have rarely been reported. To discriminate the differences of these CRP cultivars, herein an efficient and reliable method by combining metabolomics, DNA barcoding and electronic nose was first established. The hierarchical three-step filtering metabolomics analysis based on liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) indicated that 9 species-specific chemical markers including 6 flavanone glycosides and 3 polymethoxyflavones could be considered as marker metabolites for discrimination of the geoherb Crc from other cultivars. A total of 19 single nucleotide polymorphism (SNP) sites were found in nuclear internal transcribed spacer 2 (ITS2) of CRP, and three stable SNP sites (33, 128 and 174) in the ITS2 region can distinguish the four CRP cultivars. The electronic nose coupled with chemometrics could also be used to effectively distinguish Crc from other CRP cultivars. Therefore, our results indicated that the integrated method will be an effective strategy for discrimination of similar herbal medicines.

Simultaneous Quantification of Five Flavanone Glycosides in Rat Plasma by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry: Application to a Comparative Pharmacokinetic Study of Aurantii Fructus Immaturus and Aurantii Fructus Extracts.

Background: Aurantii Fructus Immaturus (AFI) and Aurantii Fructus (AF) are two traditional citrus herbs with health-promoting and nutritive properties. Objective: This paper presents the first attempt to simultaneously investigate the absorption of five major flavanone glycosides, namely narirutin, naringin, hesperidin, neohesperidin, and poncirin, in rat plasma following a single oral administration of AFI and AF extracts to rats. Methods: The plasma concentrations were determined by liquid-liquid extraction followed by a rapid and sensitive ultra-performance LC-tandem mass spectrometry method. Pharmacokinetic parameters were analyzed by noncompartmental modeling using DAS software. Results: The developed method was validated and successfully applied to the pharmacokinetic study of these five flavanone glycosides. Conclusions: The comparison of the pharmacokinetic parameters of flavanone glycosides showed that the absorption of AF extract was lower, while the elimination was relatively rapid, compared with those of AFI extract. Highlights: This study may be useful for further utilization of these two citrus herbs.

Evaluation of anti-lipase activity and bioactive flavonoids in the Citri Reticulatae Pericarpium from different harvest time.

BACKGROUND: Inhibition of pancreatic lipase is an attractive approach to the treatment of obesity and other metabolic disorders. Naturally occurring phytochemicals are promising sources of lipase inhibitors. PURPOSE: In the present study, the anti-lipase activity of Citri Reticulatae Pericarpium (CRP) extracts was firstly evaluated in vitro. Moreover, the dynamic alteration of bioactive flavonoids in CRP collected at different time and its correlation with anti-lipase activities was investigated. STUDY DESIGN/METHODS: Quantitative analysis of multi-components by a single-marker (QAMS) method was developed and validated for simultaneous determination of six flavonoids including narirutin, hesperidin, didymin, nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone and tangeretin. Anti-lipase activity evaluation and docking studies of the flavonoids was also carried out to screen out the candidate lipase inhibitors. RESULTS: The QAMS method validation results exhibited that the developed method had desirable specificity, linearity, precision and accuracy. CRP collected in early months contained higher concentrations of bioactive flavonoids, and exhibited more potent anti-lipase activity. CONCLUSION: Harvest timing had a significant impact on the amounts of bioactive flavonoids and the anti-lipase activities of CRP extracts. The contents of total flavonoids were positively correlated with the anti-lipase activities of CRP, and polymethoxyflavones played a significant role in the hypolipidemic effect of CRP. Nobiletin might be the most potential lipase inhibitor in CRP.

Chemicalome and metabolome profiling of polymethoxylated flavonoids in Citri Reticulatae Pericarpium based on an integrated strategy combining background subtraction and modified mass defect filter in a Microsoft Excel Platform.

Detection of metabolites in complex biological matrixes is a great challenge because of the background noise and endogenous components. Herein, we proposed an integrated strategy that combined background subtraction program and modified mass defect filter (MMDF) data mining in a Microsoft Excel platform for chemicalome and metabolome profiling of the polymethoxylated flavonoids (PMFs) in Citri Reticulatae Pericarpium (CRP). The exogenously-sourced ions were firstly filtered out by the developed Visual Basic for Applications (VBA) program incorporated in the Microsoft Office. The novel MMDF strategy was proposed for detecting both target and untarget constituents and metabolites based on narrow, well-defined mass defect ranges. The approach was validated to be powerful, and potentially useful for the metabolite identification of both single compound and homologous compound mixture. We successfully identified 30 and 31 metabolites from rat biosamples after oral administration of nobiletin and tangeretin, respectively. A total of 56 PMFs compounds were chemically characterized and 125 metabolites were captured. This work demonstrated the feasibility of the integrated approach for reliable characterization of the constituents and metabolites in herbal medicines.

Comparison of Aurantii Fructus Immaturus and Aurantii Fructus based on multiple chromatographic analysis and chemometrics methods.

To get a better understanding of the bioactive constituents in Aurantii Fructus Immaturus (AFI) and Aurantii Fructus (AF), in the present study, a comprehensive strategy integrating multiple chromatographic analysis and chemometrics methods was firstly proposed. Based on segmental monitoring, a high-performance liquid chromatography (HPLC)-variable wavelength detection method was established for simultaneous quantification of ten major flavonoids, and the quantitative data were further analyzed by hierarchical cluster analysis (HCA) and principal component analysis (PCA). A strong cation exchange-high performance liquid chromatography (SCX-HPLC) method combined with t-test and one-way analysis of variance (ANOVA) was developed to determine synephrine, the major alkaloid in AFI and AF. The essential oils were analyzed by gas chromatography-mass spectrometry (GC-MS) and further processed by partial least squares discrimination analysis (PLS-DA). The results indicated that the contents of ten flavonoids and synephrine in AFI were significantly higher than those in AF, and significant difference existed in samples from different geographical origins. Also, 9 differential volatile constituents detected could be used as chemical markers for discrimination of AFI and AF. Collectively, the proposed comprehensive analysis might be a well-acceptable strategy to evaluate the quality of traditional citrus herbs.

Comparative analysis of steroidal saponins in four Dioscoreae herbs by high performance liquid chromatography coupled with mass spectrometry.

Steroidal saponins, which exhibit multiple pharmacological effects, are the major bioactive constituents in herbal medicines from Dioscoreae species. In this study, a sensitive method based on high performance liquid chromatography-mass spectrometry (HPLC-MS) was established and validated for qualitative and quantitative analysis of steroidal saponins in four Dioscoreae herbs including Dioscoreae Nipponica Rhizome (DNR) and Dioscoreae Hypoglaucae Rhizome (DHR), Dioscoreae Spongiosae Rhizome (DSR) and Dioscoreae Rhizome (DR). A total of eleven steroidal saponins were identified by high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF/MS). Furthermore, seven major steroidal saponins was simultaneous quantified using a high performance liquid chromatography coupled with triple quadrupole mass spectrometry (HPLC-QQQ/MS). The qualitative and quantitative analysis results indicated that the chemical composition of DNR, DHR and DSR samples exhibited a high level of global similarity, while the ingredients in DR varied greatly from the other three herbs. Moreover, principal component analysis (PCA) and hierarchical clustering analysis (HCA) were performed to compare and discriminate the Dioscoreae herbs based on the quantitative data. The results demonstrated the qualitative and quantitative analysis of steroidal saponins based on HPLC-MS is a feasible method for quality control of Dioscoreae herbs.

Comprehensive profiling and characterization of quassinoids from the seeds of Brucea javanica via segment and exposure strategy coupled with modified mass defect filter.

Quassinoids, the predominant constituents in the seeds of Brucea javanica (BJ), have gained an increasing interest over the past decades since the discovery of their extensive biological activities. In the present study, a method based on the segment and exposure strategy coupled with two mass spectrometer data acquisition techniques was firstly developed and validated for comprehensive profiling of quassinoids in the seeds of BJ via high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (HPLC-QTOF/MS). The segment and exposure strategy could significantly improve the detection efficiency for trace quassinoids in the seeds of BJ. Furthermore, the five-point screening approach based on modified mass defect filter (MDF) and the visual isotopic ion technique could rapidly screen the precursor ions of interest. The fragmentation behavior of quassinoids was systematically investigated, and a total of 148 quassinoids including 86 potentially new ones were unambiguously or tentatively identified in the seeds of BJ. Collectively, our results demonstrate that the integrated strategy reported in this study has considerable potential for rapid screening of natural compounds especially for the trace ones in herbal medicines.

Diagnostic ion filtering strategy for chemical characterization of Guge Fengtong Tablet with high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

The present study was designed to characterize the chemical constituents of Guge Fengtong Tablet (GGFTT). Based on the chromatographic retention behavior, fragmentation pathways of chemical components and the published literatures, a diagnostic ion filtering strategy with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-Q-TOF/MS) was established to identify the multiple bioactive constituents of GGFTT. The rapid identification of forty-seven components, including 18 phenolic acids, 8 saponins, 14 gingerol-related compounds, and 7 diarylhepatonoids, was accomplished using this newly developed method. The coupling of HPLC-ESI-Q-TOF/MS with the diagnostic ion filtering strategy was useful and efficient for the in-depth structural elucidation of chemical compounds of GGFTT.

An integrated high resolution mass spectrometric data acquisition method for rapid screening of saponins in Panax notoginseng (Sanqi).

The aim of this study was to develop a convenient method without pretreatments for nontarget discovery of interested compounds. The segment and exposure strategy, coupled with two mass spectrometer data acquisition methods was firstly proposed for screening the saponins in extract of Panax notoginseng (Sanqi) via high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (HPLC-QTOF/MS). By gradually removing certain major or moderate interference compounds, the developed segment and exposure strategy could significantly improve the detection efficiency for trace compounds. Moreover, the newly developed five-point screening approach based on a modified mass defect filter strategy and the visual isotopic ion technique was verified to be efficient and reliable in picking out the interested precursor ions. In total, 234 ginsenosides including 67 potential new ones were characterized or tentatively identified from the extract of Sanqi. Particularly, some unusual compounds containing the branched glycosyl group or new substituted acyl groups were firstly reported. The proposed integrated strategy held a strong promise for analyses of the complex mixtures.

Metabolic profile of Guge Fengtong tablet in rat urine and bile after oral administration, using high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry.

In the present study, we developed and validated a rapid analytical method using high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (HPLC-Q-TOF/MS) to investigate the metabolic profile of Guge Fengtong tablet (GGFTT), a traditional Chinese medicine. The urine and bile samples were collected with 24 h after oral administration of GGFTT. A total of 34 compounds, including 11 parent compounds and 23 metabolites were unambiguously or tentatively identified. Our results indicated that glucuronidation, oxidation and methylation were the major metabolic pathways of the constituents in GGFTT. In addition, the results of this work also demonstrated the feasibility of HPLC-ESI-Q-TOF/MS for reliable characterization of the in vivo metabolites from herbal preparations.

Enzymatic hydrolysis-based absolute quantification of triacylglycerols in plant oil by use of a single marker.

Absolute quantification of triacylglycerols (TAGs) in plant oils is a challenge for analysts, because most of the necessary chemical standards are unavailable. In this study, a new method for absolute quantification analysis of multi-components by use of a single marker (AQAMS), using two crucial technologies, evaluation of the collection recovery without chemical standards and enzymatic hydrolysis, was used for determining the absolute content of TAGs in brucea javanica oil (BJO), using glycerol as the marker. The TAGs in BJO were initially characterized using ultrafast liquid chromatography tandem atmospheric-pressure-chemical-ionization mass spectrometry. Then the TAGs in BJO were individually collected, by target-fraction collection via high-performance liquid chromatography coupled with an evaporative-light-scattering detector (HPLC-ELSD), and their recoveries were calculated by use of a novel non-standard evaluated recovery strategy (NSER). The results revealed that the collection procedure was feasible and reliable. Finally, modified commercial TAG assay kits using glycerol as the marker were used to determine the absolute abundance of individual TAGs in the plant oils. Comparing the result with that obtained by HPLC-ELSD analysis using triolein standard, the content of triolein determined by AQAMS was closely matched. The proposed strategy is a practical measure for solving the problem of the lack of chemical standards, and provides a new method for absolute quantification in natural products of multi-components with the same backbone.