[Chemical structural features and anti-complementary activity of polysaccharide HPS1-D from Hedysarum polybotrys].

HPS1-D, an active polysaccharide,was isolated and purified from Hedysarum polybotrys. HPS1-D was obtained after treated with Savage method and H2O2, and purified with DEAE-cellulose 52 and Sephadex G-100 gel filtration chromatography. Then physicochemical property analysis, GC, methylation, partial acid hydrolysis, and NMR method were used to study chemical structural of HPS1-D. The conformation was primarily analyzed with GPC-MALLS method and Congo red reaction. The anti-complementary activity of HPS1-D was evaluated with the hemolysis assay. HPS1-D was a heteropolysaccharide and consisted of D-glucose, L-arabinose, (7.2:1.3). HPS1-D proved to be a neutral sugar, with 1, 4-and 1, 4, 6-alpha-D-glucopyranosyl residues in backbone ,and 1, 5-and 1, 3, 5-alpha-L-arabinofuranosyl residues in branches. HPS1-D has a random coil state conformation with monodisperse mass distribution in 0.9% NaCl solution. And HPS1-D had triple-helix conformation in concentrate of NaOH solution. Anti-complementary activity of HPS1-D was closed to its positive control heparin.

LIMA1 links the E3 ubiquitin ligase RNF40 to lipid metabolism.

LIMA1 is a LIM domain and Actin binding 1 protein that acts as a skeleton protein to promote cholesterol absorption, which makes it an ideal target for interfering with lipid metabolism. However, the detailed regulation of LIMA1 remains unclear. Here, we identified that ring finger protein 40 (RNF40), an E3 ubiquitin ligase previously known as an epigenetic modifier to increase H2B ubiquitination, mediated the ubiquitination of LIMA1 and thereby promoted its degradation in a proteasome-dependent manner. Fraction studies revealed that the 1-166aa fragment of LIMA1 was indispensable for the interaction with RNF40, and at least two domains of RNF40 might mediate the association of RNF40 with LIMA1. Notably, treatment with simvastatin dramatically decreased the levels of CHO and TG in control cells rather than cells with overexpressed LIMA1. Moreover, RNF40 significantly decreased lipid content, which could be reversed by LIMA1 overexpression. These findings suggest that E3 ubiquitin ligase RNF40 could directly target LIMA1 and promote its protein degradation in cytoplasm, leading to the suppression of lipid accumulation mediated by LIMA1. Collectively, this study unveils that RNF40 is a novel E3 ubiquitin ligase of LIMA1, which underpins its high therapeutic value to combat dysregulation of lipid metabolism.

[Sulfated modification and anticoagulant activity in vitro of sulfated glucan isolated from the aqueous extract of Hedysarum polybotrys].

SHG was sulfated by chlorosulfonic acid-pyridine method, and six samples which we got were prepared in different reaction conditions. There is a characteristic absorption peak near 260 nm in UV spectra and there are two characteristic absorption peaks near 1240 cm(-1) and 810 cm(-1) in the FT-IR. Degree of sulfation (DS) was calculated by elemental analysis and turbidimetry. Under the same conditions the absorption peaks become strong with the DS increase. The anticoagulant activity of SHG and sulfated modification samples was evaluated by the classic coagulant assays of prothrombin time (PT), activated partial thrombin time (APTT) live enzymes, and plasma thrombin time (TT). Results show that sulfated SHG has a good anticoagulant activity in vitro, and DS increased activity within a certain range.

[Investigation on chromatogram-pharmacodynamics relationship of Angelica sinensis on effect of replenishing blood].

Blood deficiency model of mice was copied by subcutaneous injection with 200, 100 and 100 mg x kg(-1) (0.01 mL x g(-1)) acetyl phenylhydrazine (APH) at the frist, fourth, and seventh days. Mice in each group were perfused with different extracted parts of Angelica sinensis (drug dosage was 2.4 g x kg(-1)) at the tenth day, once a day for 10 days. Then compare the influence of different extracted parts of Angelica sinensis to RBC, Hb, PLT and thymus, spleen and weight changes of blood deficiency mice. The peak areas of each common peak from HPLC fingerprint were associated with the date of replenishing blood pharmacodynamics efficacy by using gray relation statistic, which was used to research the chromatogram-pharmacodynamics relationship. The results showed that the part of DSC has the better effect in replenishing blood. The contribution degree of the DSC to replenishing blood of each component were determined by correlation size, and ferulic acid made the largest contribution, but contribution of other components should not be ignored. In this paper, we research the relationship of the HPLC fingerprint and spectrum activity relationship, determine the material basis of the DSC for replenishing blood, and provide effective way to represent the spectral correlation effect.

The role of long non-coding RNA LINC01296 in oral squamous cell carcinoma: a study based on bioinformatics analysis and in vitro validation.

Background: Oral squamous cell carcinoma (OSCC) is a common malignancy in the oral cavity that represents a significant global health problem. Multivariate analysis has shown that long non-coding RNA LINC01296 plays an important role in cancer biology. However, the functions of LINC01296 in OSCC are still unknown. Methods: The RNA profiles and clinicopathological information of OSCC patients and healthy subjects were downloaded. The expression level and prognostic value of LINC01296 were assessed. The functions and pathways of LINC01296were explored using the Gene Set Enrichment Analysis (GSEA) and functional analysis. The expression of LINC01296 in OSCC tissues and cell lines was determined by RT-qPCR. MTS assay was used to evaluate cell growth. The migration and invasion capacities of cells were assessed by wound healing assay and Transwell assay. Results: LINC01296 was overexpressed in the TCGA-OSCC datasets. High LINC01296expression was strongly correlated with poor outcomes of OSCC patients. LINC01296 was overexpressed in OSCC tissues compared with para-carcinoma tissues. Moreover, the expression of linc01296 was higher in CAL-27, HSC-2, and SCC-25 cells than in normal human oral keratinocytes (NHOKs). Functional analysis suggested that LINC01296might be involved in the regulation of the Wnt and MAPK signaling pathways. Additionally, LINC01296 deficiency suppressed the growth, migration, and invasion of OSCC cells, whereas overexpression of TFAP2A-AS1 cause opposite results. Conclusion: Our study showed that LINC01296 promoted OSCC cell growth, migration, and invasion, suggesting that LINC01296 might be a potential therapeutic target for OSCC.

Development and Validation of a Rapid and Simple UPLC-ESI-MS Method for Pharmacokinetics and Tissue Distribution of Astragaloside III in Rats.

A rapid and simple ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) method for the determination of astragaloside III was developed and used in a pharmacokinetic and tissue distribution study in rats following the oral administration 95% ethanol extraction of Zhenqi Fuzheng capsules. Although astragaloside III and astragaloside IV have the same molecular weight and very similar structures, they were successfully separated using this method. Quantification was performed using low-energy collision tandem mass spectrometry (CID-MS-MS) with the multiple reaction monitoring scan mode of the following precursor ion → product ion atm/z807.61→335.22 for astragaloside III and atm/z633.18→331.18 for the internal standard (hesperidin). Both astragaloside III and astragaloside IV in rat plasma were best fit to a two-compartment model. The tissue distribution study showed the overall trend of disposition of astragaloside III were C thymus > C spleen > C stomach > C liver > C heart > C kidney > C lung > C testicle The high levels of astragaloside III in thymus and spleen indicated an accumulation in organs involved in immune responses and showed that these organs are major target sites in vivo The results in the article will provide valuable information for use in clinical applications of astragaloside III.

Simultaneous determination of 10 components in Bu-Zhong-Yi-Qi Wan by solid phase extraction-high performance liquid chromatography with diode array detection and evaporative light scattering detection.

An effective, accurate and reliable method for the simultaneous separation and determination of 10 major components in Chinese medicine Bu-Zhong-Yi-Qi Wan (BZYQW) was developed and validated using solid phase extraction column-high performance liquid chromatography-diode array detection-evaporative light scattering detection (SPE-HPLC-DAD-ELSD). The chromatographic separation was performed on a Spursil™ C18 column (250 mm × 4.6 mm, 5 µm) at 30°C with an acetonitrile-water gradient as mobile phase. The DAD detection wavelength 254 nm was utilized for the quantitative analysis. The drift tube temperature and the carrier gas flow rate of the ELSD detection was set at 110.5°C and 3.1 mL/min. The total run time is 103 min, these determined components peak out within 81 min. Excellent linear behaviors over the investigated concentration ranges were observed with the values of r(2) higher than 0.9990 for all the analytes. The Linear range over hesperidin, senkyunolide I, senkyunolide H, ononin, calycosin, formononetin, ligustilide, butylene phthalide, astragaloside IV, astragaloside I is 4.50-94.50 µg/mL, 22.75-364.00 µg/mL, 2.30-45.00 µg/mL, 11.76-125.14 µg/mL, 4.62-50.35 µg/mL, 1.90-28.93 µg/mL, 1.29-159.00 µg/mL, 2.90-36.00 µg/mL, 35.40-192.40 µg/mL, 41.40-96.60 µg/mL, respectively. The method was validated by its repeatability [relative standard deviation (RSD) < 3.54%] and intra-day (RSD < 2.11%) and inter-day precision (RSD < 3.45%). The limits of detection and quantification of each component were in the ranges of 0.04-10.24 and 0.12-39.22 µg/mL, respectively. The average recovery yields of the 10 compounds ranged from 95.79 to 101.25%. The validated method was successfully applied to the simultaneous determination of these principal components in 10 commercial samples of BZYQW from different manufacturers.

Tissue distribution of six major bio-active components after oral administration of Zhenqi Fuzheng capsules to rats using ultra-pressure liquid chromatography-tandem mass spectrometry.

Radix Astragali (Huangqi in Chinese) and Fructus Ligustri Lucidi (Nvzhenzi in Chinese) (2:1, w/w) are combined in an herbal formulation called Zhenqi Fuzheng capsules (ZFCs) for use in China to improve immunity, promote the recovery of normal functions after surgical operations, and as the most important adjuvant therapy in cancer. In this study, the tissue distribution profiles of the six major bio-active constituents (calycosin-7-O-ß-D-glucoside, ononin, calycosin, formononetin, astragaloside IV and astragaloside II) were examined after oral administration of ZFCs to rats. All six constituents in each tissue were detected simultaneously using UPLC-ESI-MS, and the concentration of each constituent per gram of each tissue was determined. Quantification was performed using low-energy collision tandem mass spectrometry (CID-MS/MS) in multiple reaction monitoring (MRM) scan mode for the following precursor ion→product ion transitions at m/z 447.21→285.30 for calycosin-7-O-ß-D-glucoside, m/z 285.29→270.38 for calycosin, m/z 431→269 for ononin, m/z 269→237 for formononetin, m/z 807.40→627.50 for astragaloside IV, m/z 849.60→669.65 for astragaloside II and m/z 633.18→331.18 for the internal standard (hesperidin). The results showed that in general the tissue concentrations for all six constituents were in the following order: spleen>stomach>thymus>lung>liver>kidney>heart>testicle. The high levels in the spleen and thymus indicated that all six compounds accumulated in organs involved in the immune response, consistent with the immunity effects of ZFC. The high levels in the stomach were consistent with the oral administration of ZFC. This study was the first to compare the tissue distribution of calycosin-7-O-ß-D-glucoside with that of calycosin or of ononin with that of formononetin in rats. It was also the first study to examine the tissue distribution of astragaloside II, calycosin and formononetin following oral administration of ZFC to rats.

Simultaneous determination of calycosin-7-O-ß-D-glucoside, ononin, calycosin, formononetin, astragaloside IV, and astragaloside II in rat plasma after oral administration of Radix Astragali extraction for their pharmacokinetic studies by ultra-pressure liquid chromatography with tandem mass spectrometry.

A sensitive and reliable ultra-pressure liquid chromatography with tandem mass spectrometry (UPLC-MS) was developed and validated for simultaneous quantification of six main bioactive components, i.e., calycosin-7-O-ß-D-glucoside, ononin, calycosin, formononetin, astragaloside IV, and astragaloside II in rat plasma after oral administration of the 95 % ethanol extraction from Radix Astragali. Plasma samples were extracted with Waters Oasis(TM) HLB 1 cc (30 mg) Extraction Cartridges (SPE) separated on an UPLC™ BEH C18 column and detected by MS with electro spray ionization interface in positive selective ion monitoring mode. Calibration curves offered linear ranges of two orders of magnitude with r (2) > 0.99. The method had the lower limit quantification of 1.30, 0.73, 1.17, 2.33, 0.63, and 0.83 ng/mL for ononin, calycosin, calycosin-7-O-ß-D-glucoside, formononetin, astragaloside IV, and astragaloside II, respectively, with precision less than 10 %. The RSD of intra- and inter-day variations ranged from 1.66 to 6.46 and 3.39 to 6.58 %. This developed method was applied subsequently to pharmacokinetic studies of the six compounds in rats successfully. The proposed method was for the first time to compare the pharmacokinetic difference between calycosin-7-O-ß-D-glucoside and calycosin in rat plasma, so as between ononin and formononetin, and studied to the astragaloside II pharmacokinetics in rat plasma.

Component analysis and structure identification of active substances for anti-gastric ulcer effects in Radix Astragali by liquid chromatography and tandem mass spectrometry.

This study provided a comprehensive component analysis and structure identification of active substances for the anti-gastric ulcer effects of Radix Astragali. The data were generated by organically combining the results from in vivo pharmacodynamic experiments, a cell growth-promoting assay, structure identification, content determination, fingerprinting, and correlation analyses. The fingerprints from high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) and from HPLC coupled with evaporative light scattering detectors (ELSD) from 95% ethanol extracts of Radix Astragali (ERA) were determined using HPLC-DAD-ELSD. The structures of 16 compounds were identified using ultra-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS). The contents of these 16 compounds were simultaneously determined in a single run using HPLC-DAD-ELSD. The strength of the anti-ulceration effect of each of the 16 compounds was correlated to its content in the HPLC spectrum using gray relation statistics. The sequence of the contribution from each of the 16 compounds to the anti-gastric ulcer effect was determined. The results showed that ononin, astragalosideIII, and astragalosideIV contributed most to the observed anti-gastric ulcer effects and that these three compounds also exhibited strong growth-promoting effects in cultured GES-1 cells. The results of this study can be used to evaluate the quality of Radix Astragali and to provide a theoretical foundation for its further study.