Yinchen gongying decoction mitigates CCl4-induced chronic liver injury and fibrosis in mice implicated in inhibition of the FoxO1/TGF-ß1/ Smad2/3 and YAP signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver fibrosis (LF) is a common reversible consequence of chronic liver damage with limited therapeutic options. Yinchen Gongying decoction (YGD) composed of two homologous plants: (Artemisia capillaris Thunb, Taraxacum monochlamydeum Hand.-Mazz.), has a traditionally application as a medicinal diet for acute icteric hepatitis. However, its impact on LF and underlying mechanisms remain unclear. AIM OF THE STUDY: This study aims to assess the impact of YGD on a carbon tetrachloride (CCl4) induced liver fibrosis and elucidate its possible mechanisms. The study seeks to establish an experimental foundation for YGD as a candidate drug for hepatic fibrosis. MATERIALS AND METHODS: LC-MS/MS identified 11 blood-entry components in YGD, and network pharmacology predicted their involvement in the FoxO signaling pathway, insulin resistance, and PI3K-AKT signaling pathway. Using a CCl4-induced LF mouse model, YGD's protective effects were evaluated in comparison to a positive control and a normal group. The underlying mechanisms were explored through the assessments of hepatic stellate cells (HSCs) activation, fibrotic signaling, and inflammation. RESULTS: YGD treatment significantly improved liver function, enhanced liver morphology, and reduced liver collagen deposition in CCl4-induced LF mice. Mechanistically, YGD inhibited HSC activation, elevated MMPs/TIMP1 ratios, suppressed the FoxO1/TGF-ß1/Smad2/3 and YAP pathways, and exhibited anti-inflammatory and antioxidant effects. Notably, YGD improved the insulin signaling pathway. CONCLUSION: YGD mitigates LF in mice by modulating fibrotic and inflammatory pathways, enhancing antioxidant responses, and specifically inhibiting FoxO1/TGF-ß1/Smad2/3 and YAP signal pathways.

Anthocyanins Prevent AAPH-Induced Steroidogenesis Disorder in Leydig Cells by Counteracting Oxidative Stress and StAR Abnormal Expression in a Structure-Dependent Manner.

Testosterone deficiency may increase the risk of sexual dysfunction and the failure of spermatogenesis. Oxidative stress that is derived from the destruction of homeostasis, disease, and exposure to contaminants can damage the steroidogenicity process in Leydig cells, resulting in a reduction in testosterone synthesis. Anthocyanins are a group of innoxious antioxidants widely recognized in food sources, and are an ideal candidate to relieve oxidative stress-related steroidogenesis disorder. However, there is still a major gap in our knowledge of the structure-function relationship of anthocyanin on the activity mentioned above. In the present study, four anthocyanins including cyanidin-3-glucoside (Cy-3-glu), delphinidin-3-glucoside (Dp-3-glu), pelargonidin-3-glucoside (Pg-3-glu), and cyanidin-3,5-diglucoside (Cy-3,5-diglu) were applied to reverse testosterone generation after employing 2,2'-Azobis(2-amidinopropane)-dihydrochloride (AAPH) as the inducer of oxidative stress in R2C cells. The results demonstrated that all four kinds of anthocyanins can inhibit ROS generation, alleviate mitochondrial membrane potential damage, and contribute to increased testosterone. Among them, Cy-3,5-diglu with diglycoside performed best on antioxidative ability and improved cell dysfunction and upregulated the expression of the steroidogenic acute regulatory protein (StAR). The molecular docking further revealed the direct combination between anthocyanins and StAR, suggesting that anthocyanins with monosaccharide were more likely to interact with StAR than with diglycoside. Taken together, these data indicate that recipient R2C cells under oxidative stress submitted to anthocyanins exhibited improved steroidogenesis in a structure-dependent manner. Anthocyanins could be considered the ideal ingredients against oxidative stress-induced testosterone deficiency.

Effect of extra-column volume on practical chromatographic parameters of sub-2-µm particle-packed columns in ultra-high pressure liquid chromatography.

Effects of extra-column volume on apparent separation parameters were studied in ultra-high pressure liquid chromatography with columns and inlet connection tubings of various internal diameters (id) using 50-mm long columns packed with 1.8-µm particles under isocratic conditions. The results showed that apparent retention factors were on average 5, 11, 18, and 41% lower than those corrected with extra-column volumes for 4.6-, 3.0-, 2.1-, and 1.0-mm id columns, respectively, when the extra-column volume (11.3 µL) was kept constant. Also, apparent pressures were 31, 16, 12, and 10% higher than those corrected with pressures from extra-column volumes for 4.6-, 3.0-, 2.1-, and 1.0-mm id columns at the respective optimum flow rate for a typical ultra-high pressure liquid chromatography system. The loss in apparent efficiency increased dramatically from 4.6- to 3.0- to 2.1- to 1.0-mm id columns, less significantly as retention factors increased. The column efficiency was significantly improved as the inlet tubing id was decreased for a given column. The results suggest that maximum ratio of extra-column volume to column void volume should be approximately 1:10 for column porosity more than 0.6 and a retention factor more than 5, where 80% or higher of theoretically predicted efficiency could be achieved.

[Relationship between fibrinogen Bß-148C/T polymorphism and coronary artery lesions in children with Kawasaki disease].

OBJECTIVE: To study the possible relationship between coronary artery lesions and fibrinogen Bbeta-148 C/T polymorphism in children with Kawasaki disease. METHODS: Fast blood samples were taken from 36 children with Kawasaki disease (21 had coronary artery lesions) and 49 age- and gender-matched healthy children (control group). Plasma levels and molecular reactivity of fibrinogen were measured with Assist Plasma Fibrinogen Activity Assay System. Polymerize chain reaction and restriction enzyme digestion were used to detect the genotypes of fibrinogen Bbeta-148C/T gene polymorphism. RESULTS: The plasma fibrinogen levels in patients with coronary artery lesions were significantly higher than those in patients without coronary artery lesions and in the control group. T allele frequency in patients with Kawasaki disease was significantly higher than that in the control group. The patients with coronary artery lesions had more increased T allele frequency compared with the patients without coronary artery lesions. CONCLUSIONS: Plasma fibrinogen levels and fibrinogen Bbeta-148C/T polymorphism are associated with coronary artery lesions in children with Kawasaki disease.

Microscale HPLC enables a new paradigm for commercialization of complex chiral stationary phases.

The small column size (0.3 mm i.d. x 15 cm) used in microscale HPLC contains only a small fraction (<1%) of the chromatographic packing material of a typical analytical HPLC column. Consequently, chromatographic stationary phases that are prohibitively expensive in conventional HPLC, owing either to synthetic complexity or costly starting materials, may become commercially viable in the microscale format. To illustrate this point, a previously described, synthetically complex, crown ether chiral stationary phase was prepared and evaluated in the microscale format, showing excellent separation of the enantiomers of underivatized amine analytes.

High throughput screening of active pharmaceutical ingredients by UPLC.

Ultra performance LC (UPLC) was evaluated as an efficient screening approach to facilitate method development for drug candidates. Three stationary phases were screened: C-18, phenyl, and Shield RP 18 with column dimensions of 150 mm x 2.1 mm, 1.7 microm, which should theoretically generate 35,000 plates or 175% of the typical column plate count of a conventional 250 mm x 4.6 mm, 5 microm particle column. Thirteen different active pharmaceutical ingredients (APIs) were screened using this column set with a standardized mobile-phase gradient. The UPLC method selectivity results were compared to those obtained for these compounds via methods developed through laborious trial and error screening experiments using numerous conventional HPLC mobile and stationary phases. Peak capacity was compared for columns packed with 5 microm particles and columns packed with 1.7 microm particles. The impurities screened by UPLC were confirmed by LC/MS. The results demonstrate that simple, high efficiency UPLC gradients are a feasible and productive alternative to more conventional multiparametric chromatographic screening approaches for many compounds in the early stages of drug development.

Increasing speed of enantiomeric separations using supercritical fluid chromatography.

Enantioselective separation by supercritical fluid chromatography (SFC) has been a field of great progress since the first demonstration of a chiral separation by SFC in the 1980s. The unique properties of supercritical fluids make packed column SFC the most favorable choice for fast enantiomeric separation among all of the separation techniques. In this chapter, the effect of chiral stationary phases, modifiers, and additives on enantioseparation are discussed in terms of speed and resolution in SFC. Fundamental considerations and thermodynamic aspects are also presented.

Sub-2 microm porous and nonporous particles for fast separation in reversed-phase high performance liquid chromatography.

One approach to achieve fast and efficient separations in packed column liquid chromatography (LC) is to reduce eddy diffusion and mass transfer resistance in the mobile phase using short columns packed with small particles. In this study, efficiencies of columns packed with 1.5 and 3.0 microm nonporous and porous particles were compared in reversed-phase LC using nitromethane and a protein as analytes. Nonporous particles provided overall higher efficiency at high linear velocities when nitromethane was used as solute; however, the efficiency difference diminished significantly when the particle size was reduced from 3 to 1.5 microm. Efficiencies for 1.5 microm nonporous particles were considerably higher than those for 1.5 microm porous particles at high linear velocities when a protein, alpha-chymotrypsinogen A (MW 25,000), was used as solute. In addition, the average retention factor for amylbenzene in a column packed with ACQUITY C(18) porous particles was approximately 16 fold higher than for Micra C(18) nonporous particles for aqueous mobile phase compositions containing from 40 to 75% acetonitrile. Pressure drop, sample loading capacity, and separation power were also evaluated and compared for porous and nonporous particles under practical conditions.

Effect of particle size on the speed and resolution of chiral separations using supercritical fluid chromatography.

Fast chiral supercritical fluid chromatography (SFC) separations have become important due to the increasing use of high-throughput experimentation (HTE) in organic synthesis. These HTE experiments can generate hundreds of samples for chiral analysis that need to be assayed in a short time. In general, chiral SFC can provide much faster analysis times compared to liquid chromatography (LC). Additionally, columns packed with smaller particles can provide faster and more efficient separations. In this study, the effect of the particle size on the speed and resolution of chiral separations by SFC was evaluated. The performance of Chiralcel OD columns packed with either 5 or 3 µm particles were compared using van Deemter or other kinetic plots. The benefits of using smaller particle columns for chiral SFC analysis are illustrated.

Effect of column dimension on observed column efficiency in very high pressure liquid chromatography.

The effect of extra-column volume on observed linear velocity was investigated for columns of various internal diameters in very high pressure liquid chromatography. The results showed that the observed linear velocities were approximately 4.5, 9.5, 16.8, and 39.5% lower than the linear velocities corrected for the extra-column volume contribution for 4.6, 3.0, 2.1, and 1.0mm internal diameter columns, respectively. An empirical relationship between extra-column band broadening and extra-column volume was obtained using 50 cm long tubings of various internal diameters. The peak variance from the extra-column volume is near linearly proportional to the square of the extra-column volume for tubings with 0.0635-0.178 mm (0.025-0.07 in.) i.d. using a 50/50 acetonitrile/water mobile phase at flow rates greater than 0.3 mL/min. The effect of column internal diameter and column length on observed efficiency was studied using 50mm columns with four different column internal diameters and 2.1mm i.d columns with three different lengths. The results showed that the observed column efficiencies for 3.0, 2.1, and 1.0mm internal diameter columns were 18, 33, and 73% lower than that for a 4.6mm internal diameter column for benzophenone (k=5.5), respectively. An approximate 20% decrease in theoretical plate number was observed for propiophenone (k=3.3) using a 50 mm × 2.1 mm column packed with 1.7 µm particles compared to a 150 mm × 2.1 mm column packed with 5.0 µm particles, while the former column provided 9 fold faster separation. It is the column to extra column volume ratio instead of absolute extra-column volume that determines the degree of extra-column band-broadening in VHPLC.

Practical comparison of 2.7 microm fused-core silica particles and porous sub-2 microm particles for fast separations in pharmaceutical process development.

Fused-core silica stationary phases represent a key technological advancement in the arena of fast HPLC separations. These phases are made by fusing a 0.5 microm porous silica layer onto 1.7 microm nonporous silica cores. The reduced intra-particle flow path of the fused particles provides superior mass transfer kinetics and better performance at high mobile phase velocities, while the fused-core particles provide lower pressure than sub-2 microm particles. In this work, chromatographic performance of the fused-core particles (Ascentis Express) was investigated and compared to that of sub-2 microm porous particles (1.8 microm Zorbax Eclipse Plus C18 and 1.7 microm Acquity BEH C18). Specifically, retention, selectivity, and loading capacity were systematically compared for these two types of columns. Other chromatographic parameters such as efficiency and pressure drop were also studied. Although the fused-core column was found to provide better analyte shape selectivity, both columns had similar hydrophobic, hydrogen bonding, total ion-exchange, and acidic ion-exchange selectivities. As expected, the retention factors and sample loading capacity on the fused-core particle column were slightly lower than those for the sub-2 microm particle column. However, the most dramatic observation was that similar efficiency separations to the sub-2 microm particles could be achieved using the fused-core particles, without the expense of high column back pressure. The low pressure of the fused-core column allows fast separations to be performed routinely on a conventional LC system without significant loss in efficiency or resolution. Applications to the HPLC impurity profiling of drug substance candidates were performed using both types of columns to validate this last point.

Fundamental and practical aspects of ultrahigh pressure liquid chromatography for fast separations.

The ongoing development of HPLC has been focused on increasing the speed and efficiency of separations over the past decade. The advances in separation speed have been primarily related to the development of column technology and instrumentation. Relatively short columns packed with sub-2 microm particles provide high-speed separations while maintaining or increasing resolution. Ultrahigh pressure pump systems have been developed to overcome the high-pressure drop generated by such sub-2 microm packings. In this review, fundamental and practical aspects of ultrahigh pressure or ultrahigh performance liquid chromatography (U-HPLC) are discussed. Applications of fast U-HPLC separations are also presented.