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ObjectiveTo study the metabolism of chemical components from Citri Reticulatae Pericarpium（CRP）in different parts of rats by sequential metabolism and ultra performance liquid chromatography-high resolution mass spectrometry（UPLC-HRMS）. MethodSD male rats were employed as experimental subjects， and blood samples of intestinal metabolism and hepatic metabolism were prepared after administration of CRP ethanol extract by in situ intestinal perfusion， and comprehensive metabolic samples were collected after intragastric administration. UPLC-HRMS was used to analyze the samples with acetonitrile（A）-0.1% formic acid aqueous solution（B）as the mobile phase for gradient elution（0-10 min， 10%-30%A； 10-30 min， 30%-95%A； 30-31 min， 95%-10%A； 31-35 min， 10%A）at a flow rate of 0.35 mL·min-1， using a heated electrospray ionization with positive and negative ion mode scanning in the range of m/z 100-1 500. Under these conditions， the differences in the profiles of CRP ethanol extract， blank plasma and drug-containing plasma under different treatment groups were compared， and the chemical components of each sample were analyzed and identified based on the retention time， accurate relative molecular mass， primary and secondary ion fragments， and the information of reference substances. ResultA total of 44 chemical components were identified in the CRP ethanol extract， including flavone-O-glycosides， flavone-C-glycosides and polymethoxyflavonoids， etc. The results of sequential metabolism showed that 22 chemical components in CRP were detected in the intestinal metabolic sample， 18 chemical components were detected in the hepatic metabolic sample， and 9 identical chemical components（narirutin， hesperidin， meranzin， 5，7，8，3ʹ，4ʹ，5ʹ-hexamethoxy-flavone， isosinensetin， sinensetin， 3，5，6，7，8，3ʹ，4ʹ-heptamethoxyflavone， nobiletin and tangeretin）could be detected in all three metabolic samples， with a total of 22 compounds entering the blood in prototype form. ConclusionThe identified 21 components with well-defined structures entering the blood as prototypes may be potential active components of CRP， and differences in the components at different metabolic parts can provide an experimental basis for elucidating the in vivo biotransformation process of the metabolic components of CRP.
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Objective:To analyze the chemical constituents in Microctis Folium by ultra performance liquid chromatography-quadrupole-time-of-flight high resolution mass spectrometry （UPLC-Q-TOF-MS/MS）. Method:Waters CORTECS UPLC C<sub>18</sub> column （2.1 mm×150 mm， 1.6 μm） was used for chromatographic separation with the mobile phase of methanol （A） -0.1% formic acid solution （B） for gradient elution （0-4 min， 14%-30%A； 4-16 min， 30%-58%A； 16-25 min， 58%-78%A； 25-25.1 min， 78%-98%A； 25.1-29 min， 98%A）， the flow rate was 0.25 mL· min<sup>-1</sup>， the injection volume was 1 μL. The electrospray ionization （ESI） was adopted for determining the chromatographic effluent under positive and negative ion modes， the main chromatographic peaks were assigned and distinguished by Q-TOF， and the scanning range was <italic>m</italic>/<italic>z</italic> 100-1 500. Result:A total of 31 chemical constituents in Microctis Folium were identified by confirmation of reference substances， literature comparison and high resolution mass spectrometry data analysis. The chemical constituent cluster was composed of 28 flavonoids （9 flavone C-glycosides， 10 flavonols and their glycosides， 8 proanthocyanidins， 1 xanthone） and 3 organic acids （caffeic acid， <italic>p</italic>-coumaric acid， ferulic acid）. Conclusion:UPLC-Q-TOF-MS/MS technique provides a simple， rapid and accurate method for the identification of chemical constituents in Microctis Folium. Flavone C-glycosides， flavonol oxyglycosides and proanthocyanidins are the main chemical constituents. The 7 proanthocyanidins are reported for the first time in this herb. In conclusion， the chemical profile of Microctis Folium is characterized and the findings are meaningful for the in-depth quality assessment and material basis clarification of Microctis Folium.
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Previous studies of Dendrobium officinale on anti-hypertension effect always focused only on the blood pressure,while polysaccharides of D. officinale( DOP) have been traditionally considered as one of the main effective substances. This study aimed to evaluate the effect of ethanol extract from D. officinale( DOE) on blood pressure,Glu and lipid profile in metabolic hypertensive rats induced by comprehensive dietary factors,and elucidate the composition of effective fractions from DOE. A metabolic hypertension model of rat induced by high-sugar,high-fat diet and alcohol drinking was adopted to evaluate the effect of DOE on hypertension and other metabolic disorders. Blood pressure,Glu and lipid profile were detected to find the features and differences of DOE and DOP on metabolic hypertension. Furthermore,DOE was separated with three different common solvents according to the polarity. Along with blood pressure,Glu,UA and lipid profile,hemorheology,oxidative index and aortas structure changes were adopted to evaluate the comprehensive effects of the most effective fractions on metabolic hypertension. Finally,HPLC-DAD-MS was adopted to identify the components of the most effective fraction. The SBP and Glu of models were decreased significantly after administration of DOE and DOP for 6 weeks,while TG in DOE groups also reduced dramatically. The DOE was separated with ether,n-butanol respectively and named NAF,NBF and NCF. SBP,TG,Glu,UA of model rats were decreased significantly after 4 weeks administration with NBF. The level of MDA in serum was down-regulated,while GSH-Px and T-AOC were up-regulated obviously after 12 weeks.And the blood viscosity also obviously decreased,with less collagen deposition of aortas by Masson's trichrome staining. NBF was mainly composed of phenols and flavone C-glycosides,whose aglycone was apigenin,and monosaccharide was connected to C-6 and C-8. Ethanol extract from D.officinale has an positive effect in alleviating hypertension and metabolic disorders in metabolic hypertension. Medium polarity fraction was the effective fraction of alcohol extraction from D. officinale,and mainly composed of phenols and flavone C-glycosides.
Subject(s)
Animals , Rats , Blood Pressure , Dendrobium , Ethanol , Hypertension/drug therapy , Plant Extracts/therapeutic useABSTRACT
Objective To obtain the key enzyme gene involved in flavone C-glycosides biosynthesis pathway, a flavanone 2-hydroxylase (F2H) gene was cloned from Microcos paniculata, and its bioinformatics analysis and gene expression pattern were also performed. Methods The specific primers were designed according to Unigene in F2H annotated in the transcriptome data of M. paniculata. The open reading frame (ORF) of MpF2H gene was amplified by PCR. Then the PCR product was purified and ligated to pET30a, and finally a prokaryotic expression vector pET30a-MpF2H was constructed. The bioinformation of F2H gene cDNA sequences was analyzed by some online tools. Using RT-qPCR with suitable primers, the quantitative expression analysis of MpF2H gene in different tissues, namely, buds, leaves, twigs, flowers and fruits was carried out. Results The length of MpF2H gene ORF was 1 557 bp (GenBank accession number KY652921), which encoded a protein with 518 amino acid residues, relative molecular weight of 54 500, theory isoelectric point of 5.49. In which was no transmembrane domain. It was hypothesized that this protein located in chloroplast. MpF2H gene was expressed in different tissues, with the highest expression in leaves and the lowest expression in twigs and flowers. Conclusion The expression of MpF2H gene varied widely in different tissues. The MpF2H gene was cloned from M. paniculata based on pET30a-MpF2H expression vector. This study will provide the fundamental information for the further preparation and functional research of MpF2H protein in flavone C-glycosides biosynthesis pathway.
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OBJECTIVE: To identify flavone C-glycosides in Dendrobium officinale leaves by high performance liquid chromatography with DAD and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MSn). METHODS: The chromatographic separation was carried out at 30°C on a XB C18 column (4.6 mm × 250 mm, 5 μm) eluted with gradient program. The mobile phase consisted of methanol and water containing 0.4% formic acid. The detection wavelength was 335 nm and the on-line UV spectra was recorded in the range of 190-400 nm. The mass spectra was obtained by Agilent ion trap mass spectrometer in the negative ion mode with capillary voltage 4 500 V, dry gas temperature 350°C, dry gas flow 12.0 L · min-1, nebulizer pressure 241 kPa; mass range recorded m/z 50-600. RESULTS: Eight flavone di-C-glycosides from D. officinale leaves, whose aglycone was apigenin and monosaccharide was connected with C-6 and C-8, were identified by HPLC-DAD-ESI-MSn for the first time. The study further proved the characteristics of fragmentation pattern of flavone C-glycosides in ESI-MSn. CONCLUSION: The method is simple and rapid for the identification of D. officinale leaves. The characteristics of fragmentation pattern of ESI-MSn in flavone di-C-glycosides provide a reference for rapid detection and identification of flavone C-glycosides.
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OBJECTIVE: To establish the HPLC fingerprints of flavone C-glycosides in Dendrobium officinale leaves and determine the content of apigenin-6-C-α-L-arabinoside-8-C-β-D-xyloside for the quality control. METHODS: The compounds were separated by using XB C18(4.6 mm × 250 mm, 5 μm) analytical column. The mobile phase consisted of methanol and water containing 0.4% formic acid. Gradient elution program was used. The detection wavelength was 335 nm. In total 14 batches of Dendrobium officinale leaves and 3 batches of different species from Dendrobium were analyzed. Similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine (2004AB) and principal component analysis(PCA) were used in data analysis. RESULTS: The HPLC fingerprint of flavone C-glycosides of Dendrobium officinale leaves was established. In total 9 peaks were selected as the characteristic common peaks and 8 of them were identified. There were significant differences in the fingerprint chromatograms between Dendrobium officinale leaves and different species from Dendrobium. Principal component analysis showed that apigenin-6-C-α-L-arabinoside-8-C-β-D-xyloside among the flavone di-C-glycosides was the most important component. CONCLUSION The HPLC fingerprint and content of major component can be applied for identification and quality control of Dendrobium officinale leaves.