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.

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.