LINC00630 promotes cholangiocarcinoma cell proliferation, migration and invasion by mediating the miR-199a/FGF7 axis.

Cholangiocarcinoma (CCA) is a type of cancer with a relatively low morbidity, but poor prognosis. Aberrant long non-coding RNA (lncRNA) expression has been observed in the pathological development of CCA. In the present study, lncRNA long intergenic non-protein coding RNA 630 (LINC00630) was found to be significantly upregulated in CCA tissues and cultured cells. LINC00630 expression was positively associated with histological differentiation, TNM stage and lymph node invasion. Short hairpin RNA (sh)-LINC00630 transfection could effectively decrease CCA cell proliferation, migration and invasion. Further investigations found that LINC00630 could interact with microRNA (miR)-199a, which specifically targeted fibroblast growth factor 7 (FGF7) for degradation. FGF7 overexpression restored the sh-LINC00630 transfection-induced decrease in CCA cell proliferation, migration and invasion. In conclusion, LINC00630 significantly promoted CCA cell proliferation, migration and invasion by upregulating FGF7 through miR-199a sponging.

Mechanism deconvolution of Qing Fei Pai Du decoction for treatment of Coronavirus Disease 2019 (COVID-19) by label-free integrative pharmacology assays.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) has a long history in the prevention and treatment of pandemics. The TCM formula Lung Cleansing and Detoxifying Decoction (LCDD), also known as Qing Fei Pai Du Decoction, has been demonstrated effective against Coronavirus Disease 2019 (COVID-19). AIM OF THE STUDY: This work aimed to elucidate the active ingredients, targets and pathway mechanism of LCDD related to suppression of inflammatory, immunity regulation and relaxation of airway smooth muscle for the treatment of COVID-19. MATERIALS AND METHODS: Mining chemical ingredients reported in LCDD, 144 compounds covering all herbs were selected and screened against inflammatory-, immunity- and respiratory-related GPCRs including GPR35, H1, CB2, B2, M3 and ß2-adrenoceptor receptor using a label-free integrative pharmacology method. Further, all active compounds were detected using liquid chromatography-tandem mass spectrometry, and an herb-compound-target network based on potency and content of compounds was constructed to elucidate the multi-target and synergistic effect. RESULTS: Thirteen compounds were identified as GPR35 agonists, including licochalcone B, isoliquiritigenin, etc. Licochalcone B, isoliquiritigenin and alisol A exhibited bradykinin receptor B2 antagonism activities. Atractyline and shogaol showed as a cannabinoid receptor CB2 agonist and a histamine receptor H1 antagonist, respectively. Tectorigenin and aristofone acted as muscarinic receptor M3 antagonists, while synephrine, ephedrine and pseudoephedrine were ß2-adrenoceptor agonists. Pathway deconvolution assays suggested activation of GPR35 triggered PI3K, MEK, JNK pathways and EGFR transactivation, and the activation of ß2-adrenoceptor mediated MEK and Ca2+. The herb-compound-target network analysis found that some compounds such as licochalcone B acted on multiple targets, and multiple components interacted with the same target such as GPR35, reflecting the synergistic mechanism of Chinese medicine. At the same time, some low-abundance compounds displayed high target activity, meaning its important role in LCDD for anti-COVID-19. CONCLUSIONS: This study elucidates the active ingredients, targets and pathways of LCDD. This is useful for elucidating multitarget synergistic action for its clinical therapeutic efficacy.

Computer-Aided Rapid Establishment of Fingerprint of Xiaojin Capsule by HPLC.

Traditional Chinese medicine (TCM) formulas have a significant clinical efficacy, and the fingerprint technology has been widely accepted to fully reveal the quality of TCM. Whereas, it is a great challenge to establish the fingerprint chromatogram which can fully reflect every single herb material in a short time. In this study, we used Xiaojin capsule (XJC) as a case and developed a rapid fingerprint method based on increasing the column temperature and flow rate simultaneously combined with computer-aided. First, the elution gradient was optimized based on the retention parameters and peak shape parameters of the four linear gradients, and then, the column temperature and flow rate were increased simultaneously to shorten the analysis time. Next, the standard fingerprint chromatogram of XJC, which can reflect every herb material, was generated. Finally, quality markers were screened through unsupervised cluster analysis and supervised orthogonal partial least squares discrimination analysis. Combining computer-aided with increasing column temperature and flow rate simultaneously can develop the rapid method for establishing HPLC fingerprint of XJC, which can fully reflect every single herb material and provide comprehensive quality control. The strategy for establishing HPLC fingerprint of TCM formula could be applied to other traditional Chinese medicine formulas and herbal medicine.

An offline two-dimensional supercritical fluid chromatography × reversed phase liquid chromatography tandem quadrupole time-of-flight mass spectrometry system for comprehensive gangliosides profiling in swine brain extract.

Gangliosides, widely distributed in tissues and body fluid, have been connected to the therapy of cancer and brain related diseases. The complexity of the gangliosides structures with different polar moieties coexisting, a carbohydrate moiety and a ceramide chain, make it a great challenge in separation and analysis science. This study aimed to develop a strategy on the basis of high-accuracy data collected by an offline two-dimensional (2D) supercritical fluid chromatography (SFC) × reversed phase liquid chromatography (RPLC)/quadrupole time-of-flight (Q-ToF) system, and to integrate an in-house library with self-developed software for fast screening and identification of gangliosides from a complex sample (swine brain extract). Subsequent positive-mode MS/MS was used to validate the identified gangliosides. Finally, 153 gangliosides were separated and 79 of them were identified by the in-house library and self-developed software, 4-fold more than those by manual identification (18 gangliosides). Among the identified ones, 20 were detected in swine brain for the first time. This study established an offline 2D SFC × RPLC system and provided a new method for fast screening and automatic identification of gangliosides in complex mixtures. It will be conducive to further study of biological functions of gangliosides.

Preparation of glutathione-functionalized zwitterionic silica material for efficient enrichment of sialylated N-glycopeptides.

A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry (named SiO2-DVS-GSH). The successful synthesis of the SiO2-DVS-GSH material was confirmed by FT-IR, elemental analysis, and zeta potential analysis. The effects of water content, pH value, and salt concentration in the mobile phase on the model compound (uracil, uridine, cytosine, cytidine, guanosine, xanthosine, orotic acid) retention was studied, and a hydrophilic interaction liquid chromatography (HILIC) retention feature together with electrostatic interaction of the SiO2-DVS-GSH material was observed. The prepared stationary phase was further applied for the separation of oligosaccharide. In addition, the SiO2-DVS-GSH material displayed remarkable selectivity and specificity for the sialylated N-glycopeptides' enrichment from bovine fetuin tryptic digests, even at a mass ratio of 1:1000 (w/w) to bovine serum albumin (BSA, non-glycosylated protein), showing superior performance compared to commercial ZIC-HILIC material. Moreover, the SiO2-DVS-GSH material behaved well in the N-glycopeptides' enrichment from human serum, demonstrating its promising potential for glycoproteomics of complex biological samples. Graphical Abstract A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry, and it shows remarkable selectivity and specificity for the sialylated N-glycopeptides' enrichment.

Practical method for the definition of chromatographic peak parameters in preparative liquid chromatography.

A practical method was established for the definition of chromatographic parameters in preparative liquid chromatography. The parameters contained both the peak broadening level under different amounts of sample loading and the concentration distribution of the target compound in the elution. The parameters of the peak broadening level were defined and expressed as a matrix, which consisted of sample loading, the forward broadening and the backward broadening levels. The concentration distribution of the target compound was described by the heat map of the elution profile. The most suitable stationary phase should exhibit the narrower peak broadening and it was best to broaden to both sides to compare to the peak under analytical conditions. Besides, the concentration distribution of the target compounds should be focused on the middle of the elution. The guiding principles were validated by purification of amitriptyline from the mixture of desipramine and amitriptyline. On the selected column, when the content of the impurity desipramine was lower than 0.1%, the recovery of target compound was much higher than the other columns even when the sample loading was as high as 8.03 mg/cm3 . The parameters and methods could be used for the evaluation and selection of stationary phases in preparative chromatography.

Oxidized dextran facilitated synthesis of a silica-based concanavalin a material for lectin affinity enrichment of glycoproteins/glycopeptides.

Lectin affinity chromatography (LAC) is an important enrichment technique in glycoproteomics analysis. In order to improve the effectiveness of enrichment, it is necessary to develop LAC materials with high specificity and efficiency. Herein, using oxidized dextran as the spacer, a silica-based concanavalin A material (SiO2-ODex Con A) was synthesized to enrich glycoproteins/glycopeptides. For comparison, the SiO2-Ald Con A material was synthesized using conventional (3-glycidoxypropyl) triethoxysilane (GPMS) as the initial spacer arm. The analytical merits of both Con A materials, such as non-specific adsorption, binding capacity and trapping efficiency, have been evaluated using ovalbumin. Under high performance liquid affinity chromatography (HPLAC) mode, the SiO2-ODex Con A material was highly effective in the enrichment of glycoproteins/glycopeptides attached to high-mannose-type and bi-antennary complex-type glycans. The promising potential of the SiO2-ODex Con A material was demonstrated by selective fractionation of glycoproteins from complex biological samples for glycosylation analysis.

Hydrophilic-subtraction model for the characterization and comparison of hydrophilic interaction liquid chromatography columns.

Nowadays more and more hydrophilic interaction liquid chromatography (HILIC) columns with diverse functional groups have become commercially available, which pose a challenge to select an appropriate one. However, there is no universal model to provide guidance for selecting HILIC columns. To handle this problem, a retention model named "hydrophilic-subtraction model" was developed to characterize and compare HILIC columns. The hydrophilic-subtraction model, which was designed based on the widely recognized HILIC retention mechanisms including hydrophilic partitioning, hydrogen-bonding and electrostatic interactions, was established by the retention of 41 solutes with various properties on 8 representative HILIC columns. High correlation coefficients (R(2)≥0.990) and small standard deviations (SD≤0.041) indicated that this model correlated effectively the retention with solute descriptors and column parameters. To evaluate reliability of the model, the model was further applied to characterize 15 additional HILIC columns using 41 solutes. The results of multiple linear regression confirmed the significance of the model. The regression coefficients of the model were used to investigate retention mechanisms occurring in different chromatographic systems. Based on these regression coefficients, selectivities of HILIC stationary phases were exhibited intuitively by an angle graph and a spider diagram, which could be used as guidance for researchers to select appropriate columns for HILIC separation. Additionally, a rapid and convenient procedure was proposed for characterizing HILIC columns.

A novel method for characterization and comparison of reversed-phase column selectivity.

Characterization of reverse-phase column selectivity is helpful for chromatographers to select an optimal column. A novel method, based on linear solvation energy relationships (LSERs) combined with fundamental retention equations, was developed to characterize and compare reversed-phase column selectivity. The retention times of 25 elaborately selected solutes on 12 reversed-phase columns were determined in three linear gradient elutions. Using these retention times, fundamental retention equations were acquired by a complex sample analysis software system (CSASS). When 0%, 10%, 20%, 30%, 40% and 50% acetonitrile were introduced into the fundamental retention equations, the corresponding retention factors were predicted and used to obtain LSER equations by multiple linear regression. In the gradient elution, the retention times of solutes could be accurately determined and the excessively long or short analysis time could be avoided. As the retention factor (lnkw) at a hypothetical 0% organic modifier closely reflected properties of columns, coefficients of LSERs equations obtained based on lnkw were employed to discuss the properties of different stationary phases. An angle and a spider diagram based on solvation energy vectors were used to compare selectivity differences between stationary phases, which provided a visual means for users to select appropriate columns with orthogonal or similar selectivity. These results of column selectivities were compared with those obtained by geometric orthogonality approach, and a consistent result was acquired. Finally, Click TE-CD and XCharge C18PN with highest difference in column selectivity were applied to the separation of Psoralea corylifolia extraction.

Evaluation and comparison of n-alkyl chain and polar ligand bonded stationary phases for protein separation in reversed-phase liquid chromatography.

Protein retention is very sensitive to the change of solvent composition in reversed-phase liquid chromatography for so called "on-off" mechanism, leading to difficulty in mobile phase optimization. In this study, a novel 3-chloropropyl trichlorosilane ligand bonded column was prepared for protein separation. The differences in retention characteristics between the 3-chloropropyl trichlorosilane ligand bonded column and n-alkyl chain modified (C2, C4, C8) stationary phases were elucidated by the retention equation l nk=a+cC(B). Retention parameters (a and c) of nine standard proteins with different molecular weights were calculated by using homemade software. Results showed that retention times of nine proteins were similar on four columns, but the 3-chloropropyl trichlorosilane ligand bonded column obtained the lowest retention parameter values of larger proteins. It meant that their retention behavior affected by acetonitrile concentration would be different due to lower |c| values. More specifically, protein elution windows were broader, and retentions were less sensitive to the change of acetonitrile concentration on the 3-chloropropyl trichlorosilane ligand bonded column than that on other columns. Meanwhile, the 3-chloropropyl trichlorosilane ligand bonded column displayed distinctive selectivity for some proteins. Our results indicated that stationary phase with polar ligand provided potential solutions to the "on-off" problem and optimization in protein separation.

Purification of saponins from leaves of Panax notoginseng using preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography.

Saponins are widely distributed in the plant kingdom and have been shown to be active components of many medicinal herbs. In this study, a two-dimensional purification method based on reversed-phase liquid chromatography coupled with hydrophilic interaction liquid chromatography was successfully applied to purify saponins from leaves of Panax notoginseng. Nine saponin reference standards were used to test the separation modes and columns. The standards could not be resolved using C18 columns owing to their limited polar selectivity. However, they were completely separated on a XAmide column in hydrophilic interaction liquid chromatography mode, including two pairs of standards that were coeluted on a C18 column. The elution order of the standards on the two columns was sufficiently different, with a correlation coefficient between retention times on the C18 and XAmide columns of 0.0126, indicating good column orthogonality. Therefore, the first-dimension preparation was performed on a C18 column, followed by a XAmide column that was used to separate the fractions in the second dimension. Fifty-four fractions were prepared in the first dimension, with 25 fractions rich in saponins. Eight saponins, including two pairs of isomeric saponins and one new saponin, were isolated and identified from three representative fractions. This procedure was shown to be an effective approach for the preparative isolation and purification of saponins from leaves of P. notoginseng. Moreover, this method could possibly be employed in the purification of low-content and novel active saponins from natural products.

The herbalome--an attempt to globalize Chinese herbal medicine.

The herbalome is a project with the objective of globalizing Chinese herbal medicine (CHM) by clarification of its composition, structure, and function; by establishing a standard resource library; and by interpreting the synergistic and complementary mechanisms of multi-components on multi-targets. In phase I, it focuses on the development of systematic separation methodology for resolving and analyzing the complex components in CHM and establishment of a comprehensive resource library. This review summarizes recent advances in the herbalome project with regard to innovative separation techniques and demonstration of a resource library.

Identification of diterpenoid alkaloids from the roots of Aconitum kusnezoffii Reihcb.

Three diterpenoid alkaloids, including an unreported compound, were isolated from the roots of Aconitum kusnezoffii Reichb. On the basis of spectral analysis, these three compounds were determined to be 1,15-dimethoxy-3-hydroxy-14-benzoyl-16-ketoneoline, benzoylaconine and aconitine.

Purification of compounds from Lignum Dalbergia Odorifera using two-dimensional preparative chromatography with Click oligo (ethylene glycol) and C18 column.

Purification of compounds from traditional Chinese medicines (TCMs) is an important task for understanding the chemical composition of TCMs. However, it is difficult to obtain compounds with high enough purity for identification by NMR due to the complexity of TCMs in chemical composition. In this study, a two-dimensional purification method based on a Click oligo (ethylene glycol) column and a C18 column was developed to realize an orthogonal separation in preparative level for purifying compounds efficiently. The first dimensional preparation was performed on a Click oligo (ethylene glycol) column to simplify the sample into the fractions with good separation repeatability. On the first dimension, 7.2 g sample was separated into 11 fractions with a recovery of 86% within 6 h. A C18 column was taken as the second dimension to realize the high-performance separation and rapid preparation from the fractions collected from the first dimension. Eight compounds in fraction 6 and 2 compounds in fraction 8 were isolated and identified after optimizing the separation and collection parameters. This method is a high-efficient and orthogonal preparation method to improve the separation of a complex sample and increase the purity of the compounds, which benefits from the application of novel materials in the preparation and purification.

Purification of polar compounds from Radix isatidis using conventional C18 column coupled with polar-copolymerized C18 column.

Regarding hydrophilic interaction chromatography and normal phase liquid chromatography, RPLC is another choice used to separate polar compounds with the improvement of polar-modified C18 stationary phase. In this study, a method using conventional C18 column coupled with polar-copolymerized C18 column was successfully developed for the separation and purification of polar compounds from Radix isatidis, which is one of the most commonly used traditional Chinese medicines (TCMs). An XTerra MS C18 column was used to fractionate the extract of R. isatidis and a homemade polar-copolymerized C18 column was utilized for the final purification due to its good separation selectivity and high resolution for polar compounds. The established purification system demonstrated good orthogonality for the polar compounds. As a result, ten compounds were purified and three of them were identified as 3-methyl-5-vinyloxazolidin-2-one (compound A), 5-hydroxymethyl-2-furaldehyde (compound B) and 3-methylfuran-2-carboxylic acid (compound G) based on the MS, IR and extensive NMR data, respectively. It was demonstrated to be a feasible and powerful technique for the purification of polar compounds under RPLC mode and more chemical information of TCMs will be obtained to interpret the efficiency of TCMs.

Purification of active bufadienolides from toad skin by preparative reversed-phase liquid chromatography coupled with hydrophilic interaction chromatography.

An isolation method by reversed-phase liquid chromatography coupled with hydrophilic interaction chromatography was successfully used to isolate and purify active bufadienolides from Bufo bufo gargarizans Cantor toad skin. In the first step, crude samples were run on an XTerra Prep C18 column. After screening for activity, two fractions were chosen for further purification. A Click beta-Cyclodextrin (Click-CD) column made in our laboratory was used for the second step. Seven compounds, including four stereoisomers, were obtained at high purity. The orthogonal isolation method described here was a powerful tool for isolating bufadienolides, including stereoisomers, from a natural product. This integrated method may be useful for the discovery of novel active compounds from natural products.

Selectively preparative purification of aristolochic acids and aristololactams from Aristolochia plants.

A novel method has been developed for the purification of aristolochic acids and aristololactams compounds from Aristolochia plants, a kind of typically toxic traditional Chinese medicine. In this method, Oligo (ethylene glycol) separation column which has "clustering function" for compounds in TCMs was used to produce the fractions containing the compounds with similar structures. A four-channel parallel preparative HPLC with C18 separation column was employed to purify the target compounds. The extraction sample of the blending of Radix Aristolochiae, Fructus Aristolochiae and Caulis Aristolochiae Manshuriensis was used to develop the method. Then, four aristolochic acids and three aristololactams were obtained using this method and the chemical identification was confirmed by Q-TOF-MS, (1)H NMR and (13)C NMR. Thus, this method can deal with more than one traditional Chinese medicine simultaneously. Additionally, the results demonstrated that this method was an effective way to purify target compounds selectively from TCMs.

Systematic screening and characterization of novel bufadienolides from toad skin using ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry.

During the discovery process of novel compounds, it is of significant importance to differentiate novel from known compounds in crude extracts before starting the time-consuming process of purification. Bufadienolides are the main active components of the skin of the toad Bufo bufo gargarizans Cantor (toad skin), an important traditional Chinese medicine. The fragmentation behavior and mass spectra profiles of bufadienolides standards were investigated using ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-Q-TOFMS). Several fragmentation rules were summarized and applied to characterize novel and known bufadienolides in toad skin. Characteristic substituent groups could be identified by both diagnostic ions and their relative abundance. Bufadienolide stereoisomers could be differentiated from positional isomers by comparing fragment abundance profiles. This was used to characterize new stereoisomers for known bufadienolides. A total of 39 bufadienolides were screened out using a systematic method developed in our laboratory. In addition to 19 known bufadienolides, 20 putative novel compounds, including 8 stereoisomers, were characterized. UPLC/Q-TOFMS was demonstrated to be a powerful tool for the characterization of low-abundance bufadienolides in complex samples. This study provides guidelines for the targeted isolation of novel bufadienolides from natural products.

Structure elucidation and NMR assignments for curcuminoids from the rhizomes of Curcuma longa.

Thirteen curcuminoids (1-13) were isolated from the rhizomes of Curcuma longa. Among them, 1,5-dihydroxy-1,7-bis(4-hydroxyphenyl)-4,6-heptadiene-3-one (1), 1,5-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-4,6-heptadiene-3-one (2), 1,5-dihydroxy-1-(4-hydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-4,6-heptadiene-3-one (3), and 3-hydroxy-1,7-bis-(4-hydroxyphenyl)-6-heptene-1,5-dione (4) are new compounds, and 1-(4-hydroxyphenyl)-7-(3, 4-dihydroxyphenyl)-1, 6-heptadiene-3, 5-dione (5) is isolated from natural sources for the first time. The structures of these compounds were elucidated by extensive spectroscopic analyses, especially 1D and 2D NMR spectroscopy. The (13)C NMR data and complete (1)H and (13)C NMR assignments of some known compounds are reported for the first time. In addition, the errors of (1)H and (13)C assignments reported in the literature were corrected.

Two-dimensional RPLC-RPLC system with different pH in two dimensions for separation of alkaloids from Corydalis yanhusuo W. T. Wang.

An effective method utilizing the same RP chromatographic column with different pH in first and second LC dimensions has been developed for separation of the basic compounds from traditional Chinese medicines (TCMs). In this work, the alkaloids in Corydalis yanhusuo which is an important TCM were selected as a model to develop the method. The additives and pH values of the mobile phase were optimized in this work. To investigate the feasibility of this method, off-line mode separation was performed in the experiments. According to the UV-absorption intensity, there were eight fractions collected in acidic conditions. All the fractions were analyzed in basic conditions. The results showed that the chromatographic selectivities were significantly different in the separations performed with acidic and alkaline elution systems. Complementary separation was achieved in this work. It is demonstrated that this method would be an effective tool for alkaloids research. Based on the different pH of the mobile phase in this method, it could also be suitable to analyze compounds which were sensible to the pH of the solution.