Isolation and bioactive evaluation of flavonoid glycosides from Lobelia chinensis Lour using two-dimensional liquid chromatography combined with label-free cell phenotypic assays.

Flavonoid glycosides are widespread in herbs and often used as medicines and nutraceuticals because of good bioactivities and low toxicities. However, due to their structural complexity and diversity, isolation of flavonoid glycosides and evaluation of their bioactivities are still highly challenging. To solve this problem, a new method for separation and preparation of novel flavonoid glycosides from Lobelia chinensis Lour (L. chinensis) was developed. To avoid the interference of non-flavonoids, a solid phase extraction method was used to selectively enrich the flavonoids from the total extract. Based on hydrophilic and hydrophobic properties of the flavonoid chemical structure consisting of sugar residue and diphenylpropane (C6C3C6) skeleton, a structure-guided method development strategy was employed to design a 2D-HILIC × RPLC system in the first time. After optimization of chromatographic conditions, high selectivity and symmetric peaks of flavonoids were obtained on a zwitterionic Click XIon column and a polar-modified Atlantis T3 column. Based on these two columns, a 2 D-HILIC × RPLC system was constructed and successfully enlarged from the analytical level to the preparative level. In the first dimension, 20 fractions were obtained with good peak shapes at high sample loading. In the second-dimensional preparation, nine compounds were isolated and identified. Seven of them were novel flavonoid glycosides, lobelitin A-G, and two other known compounds were linatin and diosmin, respectively. Their target activities were evaluated via label-free cell phenotypic assays. Four novel flavonoid glycosides lobelitin A-D were found to have agonistic activities at G protein-coupled receptor 35 (GPR35). These results demonstrated that this method was effective to orthogonally separate flavonoids at the preparative level, especially for novel active flavonoid glycosides. The discovery of flavonoid glycosides with novel agonistic activity on GPR35 also sheds light on the mechanisms of action of L. chinensis relevant to its clinical application.

[Preparation of macroporous silica and its application on polysaccharide derivative based chiral stationary phase].

In order to prepare 100-nm macroporous silica, spherical silica (5 µm in particle size, 10 nm in pore diameter) was treated by hydrothermal or baking methods. During hydrothermal treatment, 22 g/L NaF was added, which efficiently promoted the enlargement of pore diameters. By this method, the average pore diameter of silica reached 100 nm after heating for 48 h at 160℃ in an autoclave, but showed a poor size distribution. In the baking method, pore diameter enlargement was controlled by modifying the baking temperature, time, and the amount of double salt LiCl-NaCl added. The addition of 1.125 g LiCl·H2O and 0.75 g NaCl per 10 g silica and baking at 500℃ for 3-5 h yielded silica with 100-nm pore diameters. This method was more efficient, easier, and better in the product pore diameter distribution than hydrothermal treatment. The obtained silica was very similar to commercial Fuji-1000 gel. The macroporous silicas obtained from the hydrothermal and baking treatments were both modified with aminopropyl silane and coated by cellulose tri(3,5-dimethylphenyl carbamate) to prepare chiral stationary phases (CSPs). The CSP based on the baking-treated silica exhibited much better selectivity and resolution for enantiomers.

Qualitative and quantitative analysis of an alkaloid fraction from Piper longum L. using ultra-high performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry.

In a previous research, an alkaloid fraction and 18 alkaloid compounds were prepared from Piper longum L. by series of purification process. In this paper, a qualitative and quantitative analysis method using ultra-high performance liquid chromatography-diode array detector-mass spectrometry (UHPLC-DAD-MS) was developed to evaluate the alkaloid fraction. Qualitative analysis of the alkaloid fraction was firstly completed by UHPLC-DAD method and 18 amide alkaloid compounds were identified. A further qualitative analysis of the alkaloid fraction was accomplished by UHPLC-MS/MS method. Another 25 amide alkaloids were identified according to their characteristic ions and neutral losses. At last, a quantitative method for the alkaloid fraction was established using four marker compounds including piperine, pipernonatine, guineensine and N-isobutyl-2E,4E-octadecadienamide. After the validation of this method, the contents of above four marker compounds in the alkaloid fraction were 57.5mg/g, 65.6mg/g, 17.7mg/g and 23.9mg/g, respectively. Moreover, the relative response factors of other three compounds to piperine were calculated. A comparative study between external standard quantification and relative response factor quantification proved no remarkable difference. UHPLC-DAD-MS method was demonstrated to be a powerful tool for the characterization of the alkaloid fraction from P. longum L. and the result proved that the quality of alkaloid fraction was efficiently improved after appropriate purification.

[Fast analysis of indole alkaloids from Evodiae fructus by supercritical fluid chromatography].

A fast chromatographic separation of indole alkaloids from Evodiae fructus was developed by supercritical fluid chromatography (SFC). The initial screening of four stationary phases was investigated with a standard mixture of evodiamine and rutaecarpine, and a complex sample of indole alkaloids prepared from Evodiae fructus as probes. Later, the effects of chromatographic parameters on separation were studied including injection volume, organic modifier, additive, temperature and back pressure. The injection volume had significant impact on the peak shape. With the additives in the mobile phase, slight changes in peak shape and retention time were observed in separation. Variation in organic modifier led to dramatic change in chromatographic behavior. Both decreased temperature and increased back pressure shortened the retention time. Finally, a fast analytical method using SFC, on a Waters ACQUITY UPC2 BEH column, methanol as modifier, under 35 degrees C and 2.07 x 10(7) Pa, was developed to separate a complex sample of indole alkaloids in less than 15 min. Another rapid approach for the separation of a complex sample of indole alkaloids was developed by using ultra-high performance liquid chromatography (UHPLC). As a result, SFC can be used in the separation of natural products, giving high performance, good resolution and fast analysis speed. The difference in selectivity with UHPLC can be used to the development of natural product separation.

Alkaloids analysis using off-line two-dimensional supercritical fluid chromatography × ultra-high performance liquid chromatography.

In this study, an off-line two-dimensional (2-D) supercritical fluid chromatography (SFC) × ultra-high performance liquid chromatography (UHPLC) method with high orthogonality was developed for the analysis of the practical amide alkaloids fraction from P. longum L. The effects of SFC parameters such as column type, organic modifier, temperature and back-pressure on separation were systematically evaluated. Different selectivity was observed for different columns (BEH, BEH 2-EP, XAmide and CSH FP). An investigation was then carried out of the orthogonality of different columns and systems following a geometric approach with a set of amide alkaloid samples. The orthogonality between a CSH FP column and a BEH column reached 50.79%, which was much higher than that for the other columns. While the orthogonality between SFC and UHPLC based on an XAmide column and an HSS T3 column reached 69.84%, which was the highest of all the combinations. At last, the practical amide alkaloids fraction was analyzed with an off-line 2-D chromatography SFC × UHPLC system. In total, at least 340 peaks were detected by this method. Rapid separation in these two dimensions and easy post treatment of SFC facilitated this 2-D system for the separation of complex samples.

A novel ionic-bonded cellulose stationary phase for saccharide separation.

A hydrophilic interaction liquid chromatography (HILIC) stationary phase of cellulose-coated silica was synthesized as a novel saccharide separation material. The material, prepared with a method based on ionic interaction, was demonstrated to be efficient for immobilization of saccharides on silica supports. The method is more efficient than traditional immobilized saccharide stationary phase synthesis methods. It was evolved from a method using anion exchanger microparticles agglomerated onto macroparticles of cation exchangers to produce anion exchangers. Cationic cellulose, which has a large number of hydroxyl groups, was immobilized on sulfonated silica. The cellulose-coated stationary phase we designed used strong hydrogen bonding between cellulose hydroxyl and carbohydrate compounds for HILIC retention and separation. The stationary phase was successfully used to separate the samples of polar compounds and the complex samples of oligosaccharides, and demonstrated good reproducibility and stability. The material exhibited good separation selectivity for carbohydrates and ability to enrich glycosylated peptides. The method described here is easy to achieve, environmentally safe and innovative than other methods. It also has extensive application possibilities to separate other categories polar compounds.

Purification of amide alkaloids from Piper longum L. using preparative two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography.

A comprehensive off-line two-dimensional liquid chromatography (2D-LC) method coupling normal phase liquid chromatography (NPLC) and reversed phase liquid chromatography (RPLC) was developed for separation and purification of amide alkaloids from Piper longum L. In the first dimension, the crude alkaloid fractions were separated in NPLC mode and 20 fractions were collected. Then fractions 5-20 were selected for further purification in RPLC mode in the second dimension. The purities of RPLC fractions with similar structures were all identified accurately by ultra performance liquid chromatography (UPLC). In total, 28 compounds with high purity were obtained and their structures were comprehensively characterized by electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. The results demonstrate that this 2D NPLC × RPLC method with good orthogonality (58.3%) was effective for the preparative separation and purification of amide alkaloids from Piper longum L.

Investigation of peptoid chiral stationary phases varied in absolute configuration.

Based on chiral N-(1-phenylethyl) glycines, a series of peptoid chiral stationary phases (CSPs) terminated with achiral alkyl group (diisopropyl, t-butyl or n-butyl) and varied in absolute configuration of selector were investigated with nine axially chiral analytes. It is very interesting to observe that selectors with heterogeneous configuration form, such as S-R-R, S-R-S, S-S-R, generally performed better than S-S-S selectors. Moreover, longer S-R-S-R-S selector did not outperform the S-R-S one obviously, with several separations declined. In addition to the result that the elution order of most enantiomers depends on the asymmetric center adjacent to the terminus of selector, we presume that the moieties around this chiral center are the most important absorbing sites for chiral discrimination; the terminal group and other chiral blocks in selector may assist enantiorecognition by constructing appropriate conformation. Various terminal alkyl groups also resulted in a few differences in enantioselectivity.

Improvement of peptoid chiral stationary phases by modifying the terminal group of selector.

The role of a terminal group of a peptoid selector in a chiral separation was investigated. Six chiral stationary phases (CSPs) with the terminal groups replaced by achiral alkyl groups (n-butyl, t-butyl and diisopropyl) and chiral groups ((1R, 2R)-2-aminocyclohexyl phenylcarbamate, N'-phenyl-L-proline amide and N'-phenyl-L-leucine amide) were synthesized. Achiral terminal groups with different steric hindrances did not broaden the enantioselectivity, but improved the separation factors for some analytes, probably due to the improvement of interactions between the analytes and peptoid chain. Introducing new chiral terminal groups proved an effective way to broaden the applicable spectrum of peptoid CSPs.

Preparation of a stationary phase with quaternary ammonium embedded group for selective separation of alkaloids based on ion-exclusion interaction.

A stationary phase (named QA C10) with quaternary ammonium embedded between a propyl and a decyl chain was synthesized by immobilization of N,N-dimethyldecylamine on chloropropyl-silica surface. A set of representative neutral, basic, and acidic compounds was employed to evaluate its chromatographic properties. The results illustrated that QA C10 was a mixed-mode stationary phase possessing both hydrophobic and ionic characteristics. The QA C10 stationary phase was further used for selective separation of alkaloids from Cortex phellodendri. Under acidic condition, alkaloids could be eluted in first 8 min, while other neutral and acidic fractions were retained better on QA C10 column. Then, obtained alkaloid fraction was analyzed by LC-MS/MS and 22 alkaloids were identified. Our study confirmed the advantages and application potential of the QA C10 stationary phase for alkaloids separation.

Combination of off-line two-dimensional hydrophilic interaction liquid chromatography for polar fraction and two-dimensional hydrophilic interaction liquid chromatography×reversed-phase liquid chromatography for medium-polar fraction in a traditional Chinese medicine.

Two-dimensional liquid chromatography (2-D LC) has been widely used for the analysis of complex samples owing to its great improvement in separation selectivity and peak capacity. However, one 2-D LC system may not be enough to meet the separation requirements due to the complexity of certain samples and respective limitations of two separation modes. In this work, water extract of Scutellaria barbata D. Don, a traditional Chinese medicine, was fractionated into polar fraction and medium-polar fraction by means of solid phase extraction (SPE). The fraction preparation made it easy to select the corresponding combination of 2-D LC method from hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC). An off-line 2-D HILIC×HILIC to analyze the polar fraction and an off-line 2-D HILIC×RP-LC to analyze the medium-polar fraction were developed, respectively. In total, 749 peaks were detected: 206 peaks from the polar fraction by the 2-D HILIC×HILIC and 543 from the medium-polar fraction by the 2-D HILIC×RP-LC. The practical peak capacities obtained in both systems were 2698 and 2879, and the orthogonality reached 63.18% and 90.62%, respectively. The results demonstrated that the two systems were both highly orthogonal, and the peak capacities greatly increased.