•A surface treatment method of normal-phase thin layer chromatography (TLC) plates with dimethyl silicone oil coating.•Direct coupling of normal-phase TLC to electrostatic field induced spray ionization (EFISI)-MS analysis.•Rapid and direct identification of lipase-inhibitory alkaloids in Nelumbo nucifera leaves by the TLC-EFISI-MS method.In situ profiling compounds in complex matrices is important technology to develop in analytic chemistry. The aim of this study is to develop a direct coupling method of thin layer chromatography (TLC) to mass spectrometry (MS) via electrostatic field induced spray ionization (EFISI). We proposed a surface treatment method of normal-phase thin layer chromatography (TLC) plates with dimethyl silicone oil coating which successfully allowed TLC to couple to MS via EFISI. Different parameters affecting the ionization efficiency were investigated and optimized, including silicone oil concentrations, air-drying times, applied voltages, and TLC plate types. This optimized TLC-EFISI-MS method was successfully applied to examine lipase inhibitory components present in lotus leaves. Six active alkaloids including three aporphines and three benzylisoquinolines were profiled with their MSn (n = 4) data, or with a comparison with reference substances. This is the first report on the coupling EFISI-MS to TLC or TLC bioautography for in situ identification of active natural products.Download high-res image (163KB)Download full-size image

The kaurenoic acid-type diterpenoids in Acanthopanacis Cortex have been reported to be the major active components. However, the diterpenoids are present as position isomers that exacerbate the challenges in obtaining standards compounds. Little work has been done on the quantitative analysis of the diterpenoids in the herb. In the present study, two diterpenoid isomers ent-16βH,17-isovalerate-kauran-19-oic acid (1) and ent-16βH,17-methyl butanoate-kauran-19-oic acid (2) with high purity were separated by analytical HPLC, followed by recrystallization in acetone. Furthermore, an HPLC-ELSD method was developed and validated for simultaneous determination of 1 and 2 in 9 batches of Acanthopanacis Cortex samples. The HPLC separation and quantification was achieved in 40 min using an Agela Promosil C18 column eluted with a gradient of water and acetonitrile. The calibration curves showed good linearity (r2 ≥ 0.999 9) within the test ranges. The LOD ranged from 0.407 2 to 0.518 0 μg and LOQ ranged from 1.018 0 to 1.295 0 μg. The precisions (%RSD) were within 1.47% for the two isomers. The recovery of the assay was in the range of 98.78%–99.11% with RSD values less than 2.76%. It is the first time to establish a quantitative HPLC method for the analysis of the bioactive kaurenoic acid isomers in the herb.

AbstractIntroductionTLC bioautography for tyrosinase inhibitors has made recent progress; however, an assay with a relative low consumption of enzyme and quantitative capability would greatly advance the efficacy of related TLC bioautographic assays.ObjectiveAn improved TLC bioautographic assay for detecting tyrosinase inhibitors was developed and validated in this study.MethodsL-DOPA (better water-solubility than L-tyrosine) was used as the substrate instead of reported L-tyrosine. The effects of enzyme and substrate concentrations, reaction temperatures and times, and pH values of the reaction system as well as different plate types on the TLC bioautographic assay were optimised. The quantitative analysis was conducted by densitometric scanning of spot areas, and expressed as the relative tyrosinase inhibitory capacity (RTIC) using a positive control (kojic acid) equivalent.ResultsThe limit of detection (LOD) of this assay was 1.0 ng for kojic acid. This assay has acceptable accuracy (101.73–102.90%), intra- and inter-day, and intra- and inter-plate precisions [relative standard deviation (RSD), less than 7.0%], and ruggedness (RSD, less than 3.5%). The consumption of enzyme (75 U/mL) is relatively low. Two tyrosinase inhibitory compounds including naringenin and 1-O-β-D-glucopyranosyl-4-allylbenzene have been isolated from Rhodiola sacra guided by this TLC bioautographic assay.ConclusionOur improved assay is a relatively low-cost, sensitive, and quantitative method compared to the reported TLC bioautographic assays. Copyright © 2016 John Wiley & Sons, Ltd.

•The fragmentation patterns of twenty-one flavonol O-glycosides were studied by a UPLC-ESI-QTOF-MS/MS method.•Flavonol mono-, di-, tri- and tetra-O-glycosides were involved in this study.•Some important diagnostic fragment ions and ion pairs of flavonol O-glycosides were reported for the first time.•The proposed fragmentation behaviors have been applied to characterization of flavonol O-glycosides in Viola tianschanica.In this study, 21 flavonol O-glycoside standards including flavonol mono-, di-, tri- and tetra-O-glycosides have been systematically studied by ultra-high performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS/MS) in the negative ionization mode to analyze their fragmentation patterns. Here for the first time, the Z23− fragment (corresponding to the loss of C-2” terminal sugar moiety) was observed in MS/MS spectra of flavonol 3-O-triglycosides. The intensity ratio of [Y0-H]−/Y0− was proposed as a criterion to distinguish the interglycosidic 1 → 2 and 1 → 6 linkages in flavonol 3-O-diglycosyl-7-O-monoglycosides. The established fragmentation behaviors have been successfully applied to characterization of flavonol glycosides in Viola tianschanica. A total of 30 flavonoid glycosides including 3 flavonol mono-O-glycosides, 10 di-O-glycosides, 10 tri-O-glycosides, 4 tetra-O-glycosides and 3 flavone di-C-glycosides were identified or tentatively identified on the base of their UV profiles, MS/MS data and/or by comparing with reference substances. Among these 15 flavonoid glycosides were reported from V. tianschanica for the first time.

A high-throughput superoxide anion radical (O2•–) scavenging capacity assay based on the xanthine oxidase/xanthine reaction system was developed and validated in the present study. The reaction conditions including detection wavelength, concentrations of reactant components, reaction temperature, reaction time, pH, terminator reagent, and sample dissolving solvents were optimized. The accuracy and reliability of the assay were assessed by evaluation of linearity (r2 = 0 .9513–0.9957), precision (intraday RSD 1.13–4.05% and interday RSD 2.13–5.62%), accuracy (95.64–97.42% recovery), and stability (RSD 2.62–6.19%), as well as comparison with the conventional colorimetric method. The EC50 values obtained by the current method and the conventional assay were highly correlated (r > 0.99). This high-throughput O2•– scavenging assay may be used for screening and estimating potential superoxide anion radical (O2•–) scavengers, especially food extracts and natural products with a very small amount of test material.

A combinative method using high-speed counter-current chromatography (HSCCC) and thin layer chromatography (TLC) as an antioxidant autographic assay was developed to separate antioxidant components from the fruits of Psoralea corylifolia. Under the guidance of TLC bioautography, eight compounds including five flavonoids and three coumarins were successfully separated from the fruits of P. corylifolia by HSCCC with an optimized two-phase solvent system, n-hexane–ethyl acetate–methanol–water (1:1.1:1.3:1, v/v/v/v). The separation produced 5.91 mg psoralen, 6.26 mg isopsoralen, 3.19 mg psoralidin, 0.92 mg corylifol A, and 2.43 mg bavachinin with corresponding purities of 99.5, 99.8, 99.4, 96.4, and 99.0%, as well as three sub-fractions, in a single run from 250 mg ethyl acetate fraction of P. corylifolia extract. Following an additional clean-up step by preparative TLC, 0.4 mg 8-prenyldaidzein (purity 91.7%), 4.18 mg neobavaisoflavone (purity 97.4%) and 4.36 mg isobavachalcone (purity 96.8%) were separated from the three individual sub-fractions. The structures of the isolated compounds were identified by 1H NMR and 13C NMR. The results of antioxidant activity estimation by electron spin resonance (ESR) method showed that psoralidin was the most active antioxidant with an IC50 value of 44.7 μM. This is the first report on simultaneous separation of eight compounds from P. corylifolia by HSCCC.

Ethnopharmacological relevanceThe spikes of Prunella vulgaris have long been used as a traditional Chinese medicine to treat various inflammation-related diseases. The aim of this study was to isolate and characterize homogenous polysaccharides from this herb and to evaluate their anticomplement activity.Materials and methodsAnticomplement activity-guided fractionation of the hot water extract of P. vulgaris was performed by DEAE-cellulose and size-exclusion chromatography, yielding two homogeneous polysaccharides PW-PS1 and PW-PS2. The homogeneity, molecular weight, monosaccharide composition and linkage of the two polysaccharides were determined in addition to other chemical characterizations. The anticomplement activity of the polysaccharides was evaluated and expressed as 50% hemolytic inhibition concentration through the classical pathway (CH50 value) and alternative pathway (AP50 value). The preliminary mechanism for the complement activation cascade was also assessed.ResultsPW-PS1 and PW-PS2 were both branched acidic polysaccharides. PW-PS1 was composed of Ara, Xyl, and 4-methoxy-Glc A in a ratio of 1.0: 2.6: 0.8. The main linkages of the sugar residues of PW-PS1 included terminal β-d-Xylp, 1,4-linked β-d-Xylp, 1,3-linked α-d-Arap, 1,3,5-linked α-d-Arap, and terminal 4-methoxy-α-d-Glcp A. PW-PS2 was composed of Rha, Ara, Xyl, Gal, and Gal A in a ratio of 0.6: 1.0: 1.3: 1.8: 3.4. The main linkages between the sugar residues of PW-PS2 included terminal Araf, 1,4-linked β-d-Xylp, 1,3-linked α-d-Rhap, terminal α-d-Galp, and 1,4,6-linked α-d-Galp. PW-PS1 and PW-PS2 inhibited complement activation through both the classical and alternative pathways with CH50 values of 0.28 and 0.13 mg/mL, respectively, and AP50 values of 0.40 and 0.35 mg/mL, respectively. Preliminary mechanism studies using complement component-depleted sera showed that PW-PS1 acted on the C1q, C3, and C9 components and that PW-PS2 acted on the C1q, C2, C3, C5, and C9 components.ConclusionOur study suggested that PW-PS1 and PW-PS2 could be valuable for the treatment of diseases associated with the excessive activation of the complement system.Download high-res image (156KB)Download full-size image