| ObjectiveEuonymus alatus is the dried winged branch in Celatraceae family. Euonymus genus, was first recorded in Sheng Nong’s herbal classic and classified as middle product. Pharmacology studies have shown that it has the function of regulating immune, decreasing blood glucose, lowering blood-fat and anti-allergic. In clinical practice aspect, Euonymus alatus widespread application like diabetes,hyperlipemia, chronic kidney disease, rheumatoid arthritis and so on.We carried out research to this traditional Chinese Medicines on the material basis of anti-inflammatory bioactive compounds,aimed at discovering compounds with good active. Therefore we took studies to this plant on separating and identifying its chemical compositions systematically, as well as anti-inflammatory screening in virto. The chemical compositions and the absorbed metabolites of Chinese herbs are essential basis for exerting pharmacodynamic action. With the progress of liquid chromatography and mass spectrometry, the qualitative and quanlitative analysis of chemical compositions and absorbed components of Chinese herbs has been one of the most important means. Euonymus alatus was selected as our research subjects for chemical composition analysis and mass fragmentation rules by ultra-high-pressure liquid chromatography coupled with linear ion trap Orbitrap high resolustion mass spectrometry methods. The fragmentation pathways of flavonoids and phenols from Euonymus alatus were systematic analyzed. Besides, the quantitative methods based on UHPLC-ESI-MS/MS methods were developed and applied to study the quality and the pharmacokinetics properties of Euonymus alatus. The present study has explored new and feasible methods for material basis and the modernization research of traditional Chinese medicine.Methods1 The preliminary separation of Euonymus alatus:the 75% ethanol extract of Euonymus alatus was concentrated individually on reduced pressure until no ethanol taste to afford a residue, which was further suspended in water and partitioned successively with petroleum ether, ethyl acetate and butanol, roughly divided the polar range and get three parts.2 The activity evaluation of total extracted part and three different polar parts extracted from Euonymus alatus:we evaluated the anti-inflammation activity in vitro by the method of LPS-inducted RAW 264.7 macrophage cells were treated with four screening parts for 24h, then the NO release (inflammatory factor) was measured by the Griess reaction (nitrite reductase assay) for assessing the efficacy of anti-inflammatory.3 The air-dried and milled plant (15 kg) was extracted with 75% ethanol (each for 2 hours) by using heating reflux method. The 75% extract was concentrated under reduced pressure to afford a residue, which was further suspended in water and partitioned successively with petroleum ether, ethyl acetate and butanol to yield of the corresponding extracts, respectively. The the main anti-inflammatory fraction, including petroleum ether fraction and ethyl acetate fraction, were separated and purified by silica gel column chromatography (CC), Sephadex LH-20, macroporous resin, HPLC and some others techniques, which led to the isolation of 23 ompounds. On the basis of spectroscopic data (HR-ESI-MS,1H-NMR,13C-NMR, DEPT-NMR,1-1H COSY, HSQC and HMBC) interpretation,21 of them were identified.4 The activity evaluation of 21 compounds from Euonymus alatus. The anti-inflammation activity in vitro were evaluated by the method of LPS-inducted RAW 264.7 macrophage cells were treated with 21 compounds for 24 h, we assessed the efficacy of anti-inflammatorythe by the Griess reaction of NO release (inflammatory factor)5 The mass fragmentation patterns of flavones and phenolic acids, the main components of Euonymus alatus, were systematic studied by a LTQ-Orbitrap mass spectrometry method. Method was set as follows:the main components of Euonymus alatus, hesperidin, kaempferol, aromadendrin, quercetin, hyperoside, naringenin, catechinic acid, dehydrodicatechin A, ferulic acid, vanillic acid, chlorogenic acid and caffeic acid were selected as model components, direct infusion injected into the mass spectormetre. The sampling rate is 5 μL/min. MS full scan range was at m/z 150-1500, the high resolution full scan resolution set at 30000 (m/z 400 full width at FWHM). The MS2-MS3 of the Orbitrap high-resolution scanning scanning resolution is set to 15000, MS4 daughter ions was used LTQ dynote detection. The highest peak of one scanning was selected for further fragmentation. The fragmentation mode was the collision induced dissociation (CID), and the collision energy was 35%.Based on the setting-up of the mass spectrometry database of main constituents from Euonymus alatus and its related specis, combined with the fragmentation pattern of model components, the analysis of Euonymus alatus chemical composition were conducted by using UHPLC-LTQ-Orbitrap MSn technology. For liquid chromatography separations, a hypersil gold C18 column (2.1 mm × 100 mm,2.7 μm) was used, mobile phase consisting of acetonitrile (A) and water containing 0.1% formic acid (B), the gradient elution set as:5% A (0 min);15% A (6 min);15% A(12 min);38% A (25 min);70% A (30 min) and 90% A (35 min);90% A (40 min).Flow rate was 300 μL/min. DAD detection was ranged from 200-400 nm. Mass spectrometric conditions:electrospray ionization (ESI) was set as interface. The positive and negative ion detection mode was used, respectively. The MS scanning range was m/z 150-1500, and the ion source gas was nitrogen. High-resolution setting of full scan is 30000,15000 resolusiton was set for MS2, and 7500 for MS3 scaninng. Dynamics scanning (data-dependent) was enabled in the data dependence scan (MSn), and the highest abundance of parent ions was selected for the dynamic exclusion.6 Reviewing the present development condition of Chinese herbs Euonymus alatus, an innovative method was developed for determination of 11 representative ingredient of Euonymus alatus (hesperidin, kaempferol, aromadendrin, quercetin, hyperoside, naringenin, dehydrodicatechin A, ferulic acid, vanillic acid, chlorogenic acid and caffeic acid) by using UHPLC-ESI-MS/MS and applied to 40 samples collected in different areas and different processing. An UHPLC BEH Ci8 Waters column (2.1 mm × 50 mm,1.7 μm) was used for LC analysis. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid water system.System program was:5% A (0 min),25% A (3.5 min),90% A (7.5 min),95% A (8 min) and 95% A(10 min).Flow rate was 300 μL/min.Multiple-reaction monitoring (MRM) was employed in positive and negative mode at the same time in single analysis process. The ion pairs were as follows:ferulic acid:m/z 169â†'125;vanillic alid:m/z 167â†'152;chlorogenic acid:m/z 353â†'191;caffeic acid:m/z 179â†'135; quercetin:m/z 301â†'151; naringenin:m/z 273â†'153; aromadendrin:m/z 287â†'259; hesperidin:m/z 609â†'301; kaempferol:m/z 285â†'185; hyperoside:m/z 463â†'300; dehydrodicatechin A:m/z 675->271.The effect of origin in Euonymus alatus on the total amount of those analytes was analyzed by using SPSS software.7 A method was developed for blood drug concentration determination of the active ingredients by liquid chromatography-tandem mass spectrometry. Given the dose of 0.8 mL/100g Euonymus alatus, blood samples were obtained at 5,10,15,20,30,45,60,240, 120,480 and 720 min.The sample volume was 5 μL. Reverse-phase A Waters UHPLC BEH C18(2.1 × 100 m, 1.7 μmm) column with the column temperature at room temperature. A linear gradient elution of eluents A (acetonitrile) and B (0.1‰ acetic acid;v/v) was used for the separation. The following gradient condition was used:initial 8% A (0 min),30% A (3.5 min),85% A (7.5 min),90% A (8.5 min) and 90% A(10 min). Flow rate was set at 300 μL/min. The mass spectrometer was operated by switching the ESI source between the positive and negative modes for a single run.For structural identification of each analyte, the information-dependent acquisition (IDA) method was used to trigger the enhanced product ion (EPI) scans by analyzing MRM signals. The optimized mass transition ion-pairs (m/z) for quantitation were 169â†'125 for ferulic acid,167â†'152 for vanillic alid,353â†'191 for chlorogenic acid,287â†'259 for caffeic acid,301â†'151 for quercetin,273â†'153 for naringenin,169â†'125 for aromadendrin,609â†'301 for hesperidin,285â†'185 for kaempferol,463â†'300 for hyperoside,675â†'271 for dehydrodicatechin A and 383â†'267 for loratadine as IS. The total run time was 10.0 min.Results1 The total extract and three different polar extracts from Euonymus alatus were screened on anti-inflammatory activity. The result showed that extracts of three polarity components were all more active than total extract.2 23 compounds were isolated from the branch with phellem wing of Euonymus alatus and 21 of them were identified based on physicochemical properties and spectral data analysis. Compounds 1,2-bis(4-hydfoxy-3-methoxyphenyl)-1,3-propanediol, lyciumin, 3-oxo-21 β-H-hop-22(29)-ene, kaempferol-7-methyl ether, dihydrokaempferol-7-methyl ether, a-monopalmitin were isolated from Euonymus alatus for the first time.3 The compounds from petroleum ether fraction and ethyl acetate fraction were screened on anti-inflammatory activity. The result showed that the main compounds for anti-inflammatory activity were the compoundsl,3,5,6,7,8,9,12,15 and 17. Among them stigmast-3-one and kaempferol-7-methyl ether had the strongest anti-inflammatory activities with 10 μg/mL against dexa-methasone as the contrast.4 Based on the analysis and simulation of the key fragmentation site of eight flavonoids compounds. The key fragmentation patterns of several flavonoids with different substituents were conprehensively studied by LTQ-Orbitrap MSn and their OE fragment ions. Also, the fragmentation pathways of four important phenol compounds from Euonymus alatus, ferulic acid, vanillic acid, chlorogenic acid and caffeic acid in LTQ-Orbitrap were systematic studied.A simple and effective method was developed for characterization of flavonoids constituents and phenol compounds in the twigs of Euonymus alatus by UHPLC-LTQ-Orbitrap. Based on the proposed strategy, sixty constituents were characterized or tentatively identified from Euonymus alatus.5 We innovatively developed UHPLC-ESI-MS/MS methods for determination of eleven representative components hesperidin, kaempferol, aromadendrin, quercetin, hyperoside, naringenin, dehydrodicatechin A, ferulic acid, vanillic acid, chlorogenic acid and caffeic acid in Euonymus alatus.The correlation coefficients were all higher than 0.9993. The LODs and LOQs for each compound were less than 0.13 ng/mL and 0.67 ng/mL, which showed a high sensitivity. The overall intra- and inter-day precisions (RSD) for the investigated components were less than 3.89%. This method also was applied to evaluate the quality of 40 batches Euonymus alatus of different origin. The results demonstrated that multiple-reaction monitoring (MRM) shows effective and high sensitive advantage, which could used for quality control of Euonymus alatus.The quantitative analysis result also showed that the contents of eleven target compounds vary greatly among 40 batches of Euonymus alatus.6 Based on the UHPLC-ESI-MS/MS methods,the study on blood drug concentration determination of the eleven main components hesperidin, kaempferol, aromadendrin, quercetin, hyperoside, naringenin, dehydrodicatechin A, ferulic acid, vanillic acid, chlorogenic acid and caffeic acid after oral administration of Euonymus alatus in rats was conducted. By using the multiple-reaction monitoring (MRM) mode, the in-vivo quantitative determination of the eleven main components were developed, which showed high interference rejection ability, high sensitivity and advantage at convenience. The calibration curves were linear over the investigated concentration range:86.02-2752.60 ng/mL (vanillic acid),0.0071-0.0045 ng/mL (caffeic acid),0.59-76.08 ng/mL (ferulic acid), 0.12-7.44 ng/mL (keampferol),35.88-4593.83 ng/mL (quercetin),0.0075-3.82 ng/mL (chlorogenic acid),69.72-4462.52 ng/mL (hyperoside),1.44-91.88 ng/mL (hesperidin), 28.19-7218.86 ng/mL (naringenin),75.68-2421.89 ng/mL (dehydrodicatechin A) and 0.23-14.58 ng/mL(aromadendrin), with all correlation coefficients higher than 0.9993. The lower limits of quantitation (LLOQ) of these analytes were less than 0.69 ng/mL. The average extraction recoveries for all compounds were between 90.84% and 107.21%. vanillic acid, caffeic acid and ferulic acid could achieve the maximum plasma concentration at 0.5 h, while keampferol, quercetin, hesperidin, naringenin, aromadendrin and chlorogenic acid could achieve the maximum plasma concentration at 1 h after oral administration. But hyperoside and dehydrodicatechin A need 2 h to achieve the maximum plasma concentration after oral administration.ConclusionIn this paper, we selected one clinical popular TCM herbs:Euonymus alatus as the objects to launch a series of researches on pharmaceutical foundation. These studies included the base of principal active components and chemical constituents, the chemical constituents of anti-inflammatory bioactivities, the the method of quality control standards researches, which were by using ultra high pressure liquid chromatography coupled with high resolustion mass spectrometry or tandem mass spectrometry. The present study reveals the chemical foundation of Euonymus alatus, provides methodological reference for the on-line rapid detection of the Chinese traditional medicine in vivo and in vitro composition, and further explore feasible technology and method for the modernization of Chinese medicine. |
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