A UPLC-MS/MS coupled with GC-MS method for quantification of twenty-one chemical ingredients from Suxiao Jiuxin pill in multiple tissue of rat and its application to tissue distribution study.

Suxiao Jiuxin pill (SJP) was a commonly-used traditional Chinese medicine for treating cardiovascular diseases. It was composed of the rhizome of Ligusticum chuanxiong Hort. and Borneolum Syntheticum. The distribution of SJP in vivo was still ambiguous. A UPLC-MS/MS coupled with GC-MS method was developed to quantify twenty-one chemical ingredients in multiple tissues from rat after administration of SJP. Protein precipitation and liquid-liquid microextraction were both utilized in sample pretreatment. All analytes were detected under acceptable specificity, linearity (correlation coefficient > 0.992), sensitivity (LLOQ < 12.5 ng/mL), precision (RSD < 14.8 %), accuracy (RE < ±14.6 %), extraction recovery (between 52.8 % and 124.1 %), matrix effect (ranged from 60.5 % and 149.7 %) and stability (RE < ±16.0 %). The established method was successfully applied in the tissue distribution study of SJP in rats. As a result, the distribution characteristics of ten analytes were clearly elucidated, including borneol, isoborneol, ligustilide, senkyunolide A, ferulic acid, senkyunolide I, levistolide A, neocnidilide, senkyunolide H and angelicide. The information provided by this research was greatly meaningful for the active chemical ingredient exploration and clinical application of SJP.

Integrated metabolomic and transcriptomic analysis reveals the effects and mechanisms of Jinqi Jiangtang tablets on type 2 diabetes.

BACKGROUND: Type 2 diabetes (T2DM) is one of the major metabolic diseases and poses a serious challenge to human life and global economic development. Jinqi Jiangtang Tablets (JQJT) is effective in ameliorating the effects of T2DM, but the mechanism of JQJT is unclear. PURPOSE: This study integrated metabolomics and transcriptomics to reveal the mechanism by which JQJT improves T2DM. METHODS: The T2DM mouse model was established, and the effects of JQJT on improving T2DM were evaluated by determining the levels of blood lipids, fasting blood glucose (FBG), insulin metabolism and hepatic lipid accumulation in mice after JQJT administration for 8 weeks. Serum metabolites were detected using ultra-performance liquid chromatography/quadrupole time-of-flight-tandem mass spectrometry (UPLC-Q-TOF-MS) technology, and mouse liver differential genes were detected using transcriptomic technology. Correlation analysis was used to extract metabolites and RNA with correlations, and potential pathways were enriched and constructed using the common pathway analysis function of MetaboAnalyst 5.0. Finally, the expression of key target proteins and genes was verified by Western blot (WB) and Polymerase Chain Reaction (PCR) to further elucidate the mechanism by which JQJT improves T2DM. RESULTS: JQJT reduced FBG and lipid levels, improved insulin resistance (IR) and hepatic lipoatrophy in mice. A total of 35 differentially abundant metabolites were identified by metabolomics, and 328 differential genes were detected by transcriptomics. The integrated metabolomics and transcriptomics results suggested that JQJT may ameliorate T2DM mainly by regulating glucose and lipid metabolic pathways. WB and PCR results showed that JQJT regulates the insulin signaling pathway, involved in fatty acid metabolism, glycogen synthesis and catabolism. CONCLUSIONS: JQJT improved IR in T2DM mice by regulating the insulin signaling pathway, improving glycogen synthesis and glycolysis, and increasing hepatic triglyceride and fatty acid metabolism.

Determination of eleven components in rat plasma by UPLC-MS/MS and GC-MS for pharmacokinetic studies after oral administration of Citri Reticulatae Pericarpium extract.

Citri Reticulatae Pericarpium (CRP) is used as common health-care food and traditional Chinese medicine (TCM), which exerts pharmacological effects, such as anti-cardiovascular, anti-tumor, anti-oxidant, anti-inflammatory, anti-virus, hepatoprotective, blood pressure-lowering and neuroprotective. In this study, reliable, and sensitive ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) methods were developed and validated for the determination of eleven active components in rat plasma after oral administration of the CRP extract. The results of this method exhibited that the specificity, linearity (r > 0.999), precision and accuracy (the coefficient of variation (CV) < 11.5 %), recovery (52.9-107.9 %), matrix effects (63.8-107.5 %), and stability (CV < 10.8 %) met all requirements for the quantitation of plasma samples. The pharmacokinetic results showed that the Tmax of flavone glycosides was less than 0.7 h, and that of polymethoxyflavones and volatile components were within 1-7 h. Meanwhile, the area-under-the-curve (AUC) and concentration maximum (Cmax) of hesperidin, nobiletin, tangeretin, and D-limonene were higher than those of the other components, suggesting that the plasma exposure levels of these constituents were higher in CRP. The present research lays a foundation for elucidating the therapeutic material basis and provides a reference for further scientific research and clinical application of CRP.

Investigation of the drug-drug interaction and incompatibility mechanism between Aconitum carmichaelii Debx and Pinellia ternata (Thunb.) Breit.

ETHNOPHARMACOLOGICAL RELEVANCE: The combination of Aconitum carmichaelii Debx (Chuanwu, CW) and Pinellia ternata (Thunb.) Breit (Banxia, BX) forms an herbal pair within the eighteen incompatible medicaments (EIM), indicating that BX and CW are incompatible. However, the scientific understanding of this incompatibility mechanism, especially the corresponding drug-drug interaction (DDI), remains complex and unclear. AIM OF THE STUDY: This study aims to explain the DDI and potential incompatibility mechanism between CW and BX based on pharmacokinetics and cocktail approach. MATERIALS AND METHODS: Ultraperformance liquid chromatography-tandem mass spectrometry methods were established for pharmacokinetics and cocktail studies. To explore the DDI between BX and CW, in the pharmacokinetics study, 10 compounds were determined in rat plasma after administering CW and BX-CW herbal pair extracts. In the cocktail assay, the pharmacokinetic parameters of five probe substrates were utilized to assess the influence of BX on cytochrome P450 (CYP) isoenzyme (dapsone for CYP3A4, phenacetin for CYP1A2, dextromethorphan for CYP2D6, tolbutamide for CYP2C9, and omeprazole for CYP2C19). Finally, the DDI and incompatibility mechanism of CW and BX were integrated to explain the rationality of EIM theory. RESULTS: BX not only enhances the absorption of aconitine and benzoylaconine but also accelerates the metabolism of mesaconitine, benzoylmesaconine, songorine, and fuziline. Moreover, BX affects the activity of CYP enzymes, which regulate the metabolism of toxic compounds. CONCLUSIONS: BX altered the activity of CYP enzymes, consequently affecting the metabolism of toxic compounds from CW. This incompatibility mechanism may be related to the increased absorption of these toxic compounds in vivo.

Quantification and discovery of quality markers from Toddalia asiatica by UHPLC-MS/MS coupled with chemometrics.

INTRODUCTION: Toddalia asiatica (TA) is a classical traditional Chinese medicine used to treat rheumatoid arthritis and contusions. However, research regarding TA quality control is currently limited. OBJECTIVE: We aimed to establish a strategy for identifying quality markers that can be used for the evaluation of the quality of TA. METHOD: A rapid and efficient ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantitative determination of 19 compounds in TA from different regions. Then, the extraction process of TA was successively optimized by single-factor optimization and response surface methodology. Moreover, chemometrics was employed to confirm the correlation between quality and target compounds. RESULTS: Utilizing the UHPLC-MS/MS method, separation of the 19 bioactive compounds was achieved within 14 min. The method was validated in terms of linearity (r2 > 0.9982), precision (0.08%-3.70%), repeatability (0.50%-2.54%), stability (2.26%-5.46%), and recovery (95.8%-113%). The optimal extraction process (extraction solvent, 65% ethanol aqueous solution; solid-liquid ratio, 1:20; extraction time, 25 min) was determined with the total content of 19 bioactive compounds as indicator. Significant disparities were observed in the contents of target compounds across different batches of TA. Besides, all samples could be categorized into two distinct groups, and magnoflorine, (-)-lyoniresinol, nitidine chloride, norbraylin, skimmianine, and decarine were identified as quality markers. CONCLUSION: In the present study, we developed a strategy to improve the quality control of TA. In consideration of the pharmacodynamic activity and statistical differences, six compounds are proposed as quality markers for TA.

Simultaneous Separation and Determination of Nine Active Ingredients in Sanyetangzhiqing by Cyclodextrin-Modified Micellar Electrokinetic Capillary Electrophoresis-Diode Array Detector.

A simple and sensitive strategy using cyclodextrin-modified micellar electrokinetic chromatography with diode array detector was developed and applied for the simultaneous separation and determination of nine components in Sanyetangzhiqing (SYTZQ), a hypoglycemic and hypolipidemic agent. Several important parameters affecting separation performance were evaluated and optimized using single variable methods. Under the optimal conditions, baseline separation of the nine components, including four flavonoids (hyperoside, isoquercitrin, quercetin-3-O-glucuronoside, and astragalin), four phenolic acids (chlorogenic acid, rosmarinic acid, salvianolic acid B, and lithospermic acid), and a monoterpenoids (paeoniflorin), were achieved in less than 16 min. The correlation coefficients of the calibration curves were over 0.9996 for all the analytes. Intraday and interday precisions ranged from 0.4% to 4.8% and 1.7% to 5.0%, respectively. Recoveries of analytes varied from 95.3% to 105%. Validation results as well as the application to analyse SYTZQ samples demonstrated the applicability of the proposed method and thus provided an effective tool for the quality control of SYTZQ. Moreover, with the advantages of short time consuming, low energy consumption, high efficiency, and low cost, this method has laid a foundation for the determination and quality evaluation of multicomponents in Chinese herbal compounds.

A novel biological sources consistency evaluation method reveals high level of biodiversity within wild natural medicine: A case study of Amynthas earthworms as "Guang Dilong".

For wild natural medicine, unanticipated biodiversity as species or varieties with similar morphological characteristics and sympatric distribution may co-exist in a single batch of medical materials, which affects the efficacy and safety of clinical medication. DNA barcoding as an effective species identification tool is limited by its low sample throughput nature. In this study, combining DNA mini-barcode, DNA metabarcoding and species delimitation method, a novel biological sources consistency evaluation strategy was proposed, and high level of interspecific and intraspecific variations were observed and validated among 5376 Amynthas samples from 19 sampling points regarded as "Guang Dilong" and 25 batches of proprietary Chinese medicines. Besides Amynthas aspergillum as the authentic source, 8 other Molecular Operational Taxonomic Units (MOTUs) were elucidated. Significantly, even the subgroups within A. aspergillum revealed here differ significantly on chemical compositions and biological activity. Fortunately, this biodiversity could be controlled when the collection was limited to designated areas, as proved by 2796 "decoction pieces" samples. This batch biological identification method should be introduced as a novel concept regarding natural medicine quality control, and to offer guidelines for in-situ conservation and breeding bases construction of wild natural medicine.

Simultaneous determination of multiple components in rat plasma by UHPLC-sMRM for pharmacokinetic studies after oral administration of Qingjin Yiqi Granules.

As a Traditional Chinese Medicine prescription, Qingjin Yiqi Granules (QJYQ) provides an effective treatment for patients recovering from COVID-19. However, the pharmacokinetics characteristics of the main components of QJYQ in vivo are still unknown. An efficacious ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed and validated for the simultaneous determination of 33 components in rat plasma after oral administration of QJYQ. The plasma samples were precipitated with 400 µL methanol/acetonitrile (1/1, v/v) and analyzed in scheduled multiple reaction monitoring mode. The linear relationship of the 33 components was good (r > 0.9928). The lower limit of quantification for 33 components ranged from 0.4-60.5 ng/mL. The average recoveries and matrix effects of the analytes ranged from 72.9% to 115.0% with RSD of 1.4%-15.0%. All inter-day and intra-day RSDs were within 15.0%. After oral administration (3.15 g/kg), the validated approach was effectively applied to the pharmacokinetics of main components of QJYQ. Finally, fifteen main constituents of QJYQ with large plasma exposure were obtained, including baicalin, wogonoside, wogonin, apigenin-7-O-glucuronide, verbenalin, isoferulic acid, hesperidin, liquiritin, harpagide, protocatechuic acid, p-Coumaric acid, ferulic acid, sinapic acid, liquiritin apioside and glycyrrhizic acid. The present research lays a foundation for clarifying the therapeutic material basis of QJYQ and provides a reference for further scientific research and clinical application of QJYQ.

Gliotransmission and adenosine signaling promote axon regeneration.

How glia control axon regeneration remains incompletely understood. Here, we investigate glial regulation of regenerative ability differences of closely related Drosophila larval sensory neuron subtypes. Axotomy elicits Ca2+ signals in ensheathing glia, which activates regenerative neurons through the gliotransmitter adenosine and mounts axon regenerative programs. However, non-regenerative neurons do not respond to glial stimulation or adenosine. Such neuronal subtype-specific responses result from specific expressions of adenosine receptors in regenerative neurons. Disrupting gliotransmission impedes axon regeneration of regenerative neurons, and ectopic adenosine receptor expression in non-regenerative neurons suffices to activate regenerative programs and induce axon regeneration. Furthermore, stimulating gliotransmission or activating the mammalian ortholog of Drosophila adenosine receptors in retinal ganglion cells (RGCs) promotes axon regrowth after optic nerve crush in adult mice. Altogether, our findings demonstrate that gliotransmission orchestrates neuronal subtype-specific axon regeneration in Drosophila and suggest that targeting gliotransmission or adenosine signaling is a strategy for mammalian central nervous system repair.

A Non-Immunized and BSA-Template Aggregation-Induced Emission Sensor for Noninvasive Detection of Cystatin C in the Clinical Diagnosis of Diabetes Nephropathy.

Diabetes nephropathy (DN) is one of the main causes of death in patients with diabetes. Cystatin C (Cys C) is a reliable indicator of glomerular filtration function. Therefore, it is urgent and meaningful to obtain early warning of DN by noninvasive measurement of Cys C. In this investigation, a novel fluorescence sensor (BSA-AIEgen sensor) was synthesized by cross-linking the aggregation-induced emission (AIE) characteristics of 2-(4-bromophenyl)-3-(4-(4-(diphenylamino) styryl) phenyl) fumaronitrile (TPABDFN) and bovine serum albumin (BSA), which exhibited the "On" state owing to the restriction of the intramolecular motions (RIM) phenomenon of TPABDFN. Intriguingly, a decrease in fluorescence of BSA-AIEgen sensors could be found owing to BSA on the surface of BSA-AIEgen sensor hydrolyzed by papain, but a reverse phenomenon emerged with the increase of Cys C content as the inhibitor of papain. Hence, Cys C was successfully detected by employing the fluorescent differential display and the linear range was from 12.5 ng/mL to 800 ng/mL (R2 = 0.994) with the limit of detection (LOD) of 7.10 ng/mL (S/N = 3). Further, the developed BSA-AIEgen sensor successfully differentiates patients with diabetes nephropathy from volunteers with the advantages of high specificity, low cost, and simple operation. Accordingly, it is expected to become a non-immunized method to monitor Cys C for the early warning, noninvasive diagnosis, and drug efficacy evaluation of diabetes nephropathy.