Combined metabolomics and gut microbiome to investigate the effects and mechanisms of Yuquan Pill on type 2 diabetes in rats.

Yuquan Pill (YQP) is a traditional Chinese medicine (TCM) for the treatment of type 2 diabetes (T2DM) in China for many years, and has a beneficial clinical effect. In this study, the antidiabetic mechanism of YQP was investigated for the first time from the perspective of metabolomics and intestinal microbiota. After 28 days of high-fat feeding, rats were injected intraperitoneally with streptozotocin (STZ, 35 mg/kg) followed by a single oral administration of YQP 2.16 g/kg and metformin 200 mg/kg for 5 weeks. The results showed that YQP was effectively improved insulin resistance and alleviated hyperglycemia and hyperlipidemia associated with T2DM. YQP was found to regulate metabolism and gut microbiota in T2DM rats using untargeted metabolomics and gut microbiota integration. Forty-one metabolites and five metabolic pathways were identified, including Ascorbate and aldarate metabolism, Nicotinate and nicotinamide metabolism, Galactose metabolism, Pentose phosphate pathway and Tyrosine metabolism. YQP can regulate T2DM-induced dysbacteriosis by modulating the abundance of Firmicutes, Bacteroidetes, Ruminococcus, Lactobacillus. The restorative effects of YQP in rats with T2DM have been confirmed and provide a scientific basis for the clinical treatment of diabetic patients.

Head-to-Head Comparison of High-Performance Liquid Chromatography versus Nuclear Magnetic Resonance for the Quantitative Analysis of Carbohydrates in Yiqi Fumai Lyophilized Injection.

Carbohydrate analysis can be used as a standard analysis for quality control of industries of plants, foods and pharmaceuticals. Quantitative 1H NMR spectroscopy (qNMR) is an excellent alternative to chromatography-based mixture analysis. However, the application of qNMR in sugar analysis has rarely been reported. In this study, the performance of qNMR in sugar analysis was investigated and compared with the results from HPLC analysis. A head-to-head comparison of qNMR (internal and external standard methods) versus HPLC (PMP pre-column derivatization HPLC, HPLC-RID and HPLC-ELSD) based on quantitative analysis of four carbohydrates (fructose, glucose, sucrose and maltose) in Yiqi Fumai lyophilized injection (YQFM) is presented. Both assays showed similar performance characteristics, including linearity range, accuracy, precision and recovery, and analysis times of less than 30 min/sample. After methodological validation, both qNMR and HPLC have good accuracy, precision and stability. Indeed, the qNMR method is simple, sensitive and rapid in quantifying the four sugars. By analysis of variance (ANOVA) for sugar content with HPLC and qNMR methods, we demonstrated that the two analytical methods had no significant difference and could be used interchangeably for the quantitative analysis of carbohydrates.

Ultra-performance liquid chromatography-tandem mass spectrometry determination and pharmacokinetic studies of five phenolic acids in rat plasma after intravenous administration of salvianolic acid for injection.

A simple, efficient, and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for simultaneous determination and pharmacokinetic study of salvianolic acid D, rosmarinic acid, lithospermicic acid, salvianolic acid B, and salvianolic acid Y in rat plasma after intravenous administration of salvianolic acid for injection. Three doses of administration, containing 10, 25, and 62.5 mg/kg, were investigated. Plasma samples were pretreated using protein precipitation with pre-cooled acetonitrile. As shown in S1, Chromatographic separation was achieved on a Waters Acquity UPLC® BEH C18 column (1.7 µm, 2.1 × 100 mm) with a mobile phase composed of acetonitrile-methanol-0.5% aqueous formic acid (10:30:60, v/v/v) at a flow rate of 0.3 ml/min. MS was detected by electrospray ion source negative ion mode and multiple reaction monitoring mode. The method was fully validated. The calibration curves for the five phenolic acids were linear in the given concentration ranges. The extraction recoveries, matrix effects, intra-day and inter-day precisions, and accuracies of the five analytes were all within acceptable limits. No significant difference of elimination half-life time (T1/2 ) of five analytes at three doses was observed. Area under the curve and peak concentration (Cmax ) of the five analytes demonstrated a linear increase in the doses with the linear correlation r of each analyte at three doses being greater than 0.915. It indicated that the pharmacokinetic behavior of is positively related to the dose in the range of 10-62.5 mg/kg.

Determination of ginsenoside Rh3 in rat plasma by LC-MS/MS and its application to a pharmacokinetic study.

Ginsenoside Rh3 (GRh3) is a bacterial metabolite of ginsenoside Rg5, which is the main component of hot-processed ginseng. A simple, efficient and sensitive method was developed and validated for the determination of GRh3 in rat plasma by LC-tandem mass spectrometry. After protein precipitation with methanol/acetonitrile (1:1, vol/vol) using propranolol as the internal standard, the target analytes were separated on an XDB C18 column, with methanol containing 0.1% formic acid and water containing 0.1% formic acid used as mobile phases for gradient elution. Mass spectrometry was performed in electrospray ion source-positive ion mode and multiple reaction monitoring mode, monitoring the transitions m/z 622.5 → 425.5 and m/z 260.1 → 116.1 for GRh3 and internal standard, respectively. The concentration range of GRh3 was 20-20,000 ng/mL and the correlation coefficient (r2 ) was greater than 0.99. The accuracy error and relative standard deviation were below 15%. The extraction recovery and matrix effect were 74.2% to 78.7% and 96.9% to 108.4%, respectively. Under different conditions, GRh3 was stable in the range of 1.8%-8.7%. This method has been successfully applied to study the pharmacokinetics of GRh3 with an oral dose of 10.0 mg/kg and an intravenous dose of 2.0 mg/kg in rats, respectively. The absolute bioavailability of GRh3 was 37.6%.

Determination of Dammar-20(22)E,24-Diene-3ß,6α,12ß-Triol in rat plasma by LC-MS/MS and its application in a pharmacokinetic study.

Dammar-20(22)E,24-Diene-3ß,6α,12ß-Triol (YNPT2), as one of the main pharmacological and active components of Panax ginseng, promotes ubiquitination and degradation of hypoxia inducible factor Ia through proteasome, which reduces the content of hypoxia inducible factor Ia in tumor cells. Therefore, it is widely used in tumor inhibition. A sensitive and specific bioanalytical method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of YNPT2 rat plasma has been developed. Buspirone was used as the internal standard (IS). A 50 µl aliquot of rat plasma sample was deproteinized by 150 µl methanol-acetonitrile (1:1,v:v), vortex-mixed for 1 min and centrifuged at 15,000 r/min for 10 min at 4 °C. Then, 120 µl of supernatant was pipetted out into the autosampler vials and analyzed by LC-MS/MS with 10 µl injection volume. Chromatographic separation was performed on an Agilent ZORBAX XDB-C18 column (2.1 × 50 mm, 3.5 µm) with mobile phases consisting of water containing 5 mM ammonium acetate (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.6 ml/min over a total run time of 4.0 min. YNPT2 and buspirone (IS) were detected and quantified using positive electrospray ionization in multiple reaction monitoring (MRM) mode with transitions of m/z 441.4 â†’ 109.1 for YNPT2 and m/z 386.3 â†’ 122.1 for IS. The linear range was 5-2000 ng/ml with the square regression coefficient (r2) of 0.9972, and the lower limit of quantification (LLOQ) was 5 ng/ml. The intra-day and inter-day precision deviations of YNPT2 ranged from 3.8 to 6.9% and 3.5-5.8%, and accuracy error ranged from -7.4-5.9% and -9.2-11.9%. The average extraction recovery of YNPT2 in rat plasma was between the range of 98.5%-102.7%. This method was successfully applied to study the pharmacokinetics of YNPT2 in rats after intragastric administration at a single dose of 10.0 mg/kg and after intravenous injection at a single dose of 2.0 mg/kg.