Mechanism of XiJiaQi in the treatment of chronic heart failure: Integrated analysis by pharmacoinformatics, molecular dynamics simulation, and SPR validation.

OBJECTIVE: Chronic heart failure (CHF) is a complicated clinical syndrome with a high mortality rate. XiJiaQi (XJQ) is a traditional Chinese medicine used in the clinical treatment of CHF, but its bioactive components and their modes of action remain unknown. This study was designed to unravel the molecular mechanism of XJQ in the treatment of CHF using multiple computer-assisted and experimental methods. METHODS: Pharmacoinformatics-based methods were used to explore the active components and targets of XJQ in the treatment of CHF. ADMETlab was then utilized to evaluate the pharmacokinetic and toxicological properties of core components. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were to explore the underlying mechanism of XJQ treatment. Molecular docking, surface plasmon resonance (SPR), and molecular dynamics (MD) were employed to evaluate the binding of active components to putative targets. RESULTS: Astragaloside IV, formononetin, kirenol, darutoside, periplocin and periplocymarin were identified as core XJQ-related components, and IL6 and STAT3 were identified as core XJQ targets. ADME/T results indicated that periplocin and periplocymarin may have potential toxicity. GO and KEGG pathway analyses revealed that XJQ mainly intervenes in inflammation, apoptosis, diabetes, and atherosclerosis-related biological pathways. Molecular docking and SPR revealed that formononetin had a high affinity with IL6 and STAT3. Furthermore, MD simulation confirmed that formononetin could firmly bind to the site 2 region of IL6 and the DNA binding domain of STAT3. CONCLUSION: This study provides a mechanistic rationale for the clinical application of XJQ. Modulation of STAT3 and IL-6 by XJQ can impact CHF, further guiding research efforts into the molecular underpinnings of CHF.

Comprehensive characterization of in vitro and in vivo metabolites of 2',3',5'­tri­O­acetyl­N6­(3­hydroxyphenyl) adenosine and study of the metabolites distribution in rats by combined methods of HPLC-DAD, off-line cryoNMR, and HPLC-QTOFMS.

The compound 2',3',5'­tri­O­acetyl­N6­(3­hydroxyphenyl) adenosine (also known as IMM-H007) is a new adenosine analogue that displays anti-hyperlipidaemic activity in many preliminary studies. To clarify its biotransformation process, in vitro and in vivo metabolic patterns of IMM-H007 in rat liver microsomes (RLMs), urine, feces, serum, and various tissues were investigated using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD), off-line cryogenically cooled probe nuclear magnetic resonance (cryoNMR), and high-performance liquid chromatography quadrupole TOF MS (HPLC-QTOFMS) measurements. A total of 21 metabolites were detected and identified based on accurate mass measurements, diagnostic product ions, and 1D and 2D NMR data. All of the 21 metabolites were detected in vivo besides the 7 ones (LM1-3, LM4a-b, LM5, LM6 (M8)) in vitro. Among them, eight metabolites were phase I metabolites composed of the hydrolysis products LM1-3, LM4a, LM4b, LM5 and M7-8, and hydrolysis and hydroxylation products M6. Others were phase II metabolites including glucuronidation products M2, M4, M9, M11a-c, and M12a-c; and sulfation products M3, M5, and M10. Notably, 14 metabolites (LM1-3, LM4a-b, LM5, M9-10, M11a-c, M12a-c) were unreported before and the distribution of IMM-H007 and its all metabolites was reported for the first time. The results revealed IMM-H007 was metabolized mainly in the small intestine and serum, kidney, stomach, small and large intestines were important samples for metabolites presence. This work improves understanding of the metabolism, distribution, and excretion of IMM-H007, and demonstrates the HPLC/HPLC-MS/off-line cryoNMR approach can be applied for detection and identification of metabolites in complex biological matrices.

Beneficial Metabolic Effects of 2',3',5'-Triacetyl-N6-(3-hydroxylaniline) adenosine in Multiple Biological Matrices and Intestinal Flora of Hyperlipidemic Hamsters.

Hyperlipidemia is one of the main causes of obesity, type 2 diabetes mellitus (T2DM), and atherosclerosis. The adenosine derivative, 2',3',5'-triacetyl-N6-(3-hydroxylaniline) adenosine (IMM-H007) is an effective lipid-lowering compound that has important implications for the development of lipid-lowering drugs. Metabolomic analysis based on 1H NMR was used to monitor dynamic changes in diverse biological media including serum, liver, urine, and feces in response to high-fat diet (HFD) and IMM-H007 treatments. Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography (GC) analyses were performed to quantify the bile acids and fatty acids in the liver and feces. Fecal microbiome profiling was performed using Illumina sequencing of the 16S rRNA ( 16S rRNA) gene. IMM-H007 improved the metabolism of carbohydrate, ketone bodies, fatty acids, amino acids, and bile acids in hyperlipidemic hamsters. The correlation between metabolite changes was explored in different biological media. Significant changes in gut microbiota were observed in the HFD and IMM-H007 treatment groups. In the HFD group at the phylum level, we found high levels of the Firmicutes genus and low levels of Bacteroidetes. In contrast, the administration of IMM-H007 reversed the levels of Firmicutes and Bacteroidetes. This reversal suggested that IMM-H007 may have the ability to regulate the composition of the gut flora. We also analyzed the correlation between the gut flora and the metabolites. Our results indicate that IMM-H007 treatment improves the hyperlipidemic metabolism and the structure of the gut microbiota in hyperlipidemic hamsters.

The Application of Quantitative ¹H-NMR for the Determination of Orlistat in Tablets.

A quantitative nuclear magnetic resonance (qNMR) method to measure the content of Orlistat in tablets was studied and found to be efficient, accurate, reliable, and simple. In this paper, phloroglucinolanhydrous and dimethylsulfoxide-d6 (DMSO-d6) served as the internal standard and solvent, respectively. The qNMR methodology, including the linearity, range, the limit of detection (LOD) and quantification (LOQ), stability, precision, and accuracy, was validated seriatim, and the results were very favorable. The content determination results of three batches of Orlistat in tablets were almost identical upon comparing the qNMR method and the high-performance liquid chromatography (HPLC) method. The recommended method authentically compensated the deficiencies of the current HPLC method for determining Orlistat content, and proved to be a method complementary to traditional analysis for the purity measurement of Orlistat in some pharmaceutical preparations.

Quantitative Metabolomic Profiling of Plasma, Urine, and Liver Extracts by 1H NMR Spectroscopy Characterizes Different Stages of Atherosclerosis in Hamsters.

Atherosclerosis (AS) is a progressive disease that contributes to cardiovascular disease and shows a complex etiology, including genetic and environmental factors. To understand systemic metabolic changes and to identify potential biomarkers correlated with the occurrence and perpetuation of diet-induced AS, we applied 1H NMR-based metabolomics to detect the time-related metabolic profiles of plasma, urine, and liver extracts from male hamsters fed a high fat and high cholesterol (HFHC) diet. Conventional biochemical assays and histopathological examinations as well as protein expression analyses were performed to provide complementary information. We found that diet treatment caused obvious aortic lesions, lipid accumulation, and inflammatory infiltration in hamsters. Downregulation of proteins related to cholesterol metabolism, including hepatic SREBP2, LDL-R, CYP7A1, SR-BI, HMGCR, LCAT, and SOAT1 was detected, which elucidated the perturbation of cholesterol homeostasis during the HFHC diet challenge. Using "targeted analysis", we quantified 40 plasma, 80 urine, and 60 liver hydrophilic extract metabolites. Multivariate analyses of the identified metabolites elucidated sophisticated metabolic disturbances in multiple matrices, including energy homeostasis, intestinal microbiota functions, inflammation, and oxidative stress coupled with the metabolisms of cholesterol, fatty acids, saccharides, choline, amino acids, and nucleotides. For the first time, our results demonstrate a time-dependent metabolic progression of multiple biological matrices in hamsters from physiological status to early AS and further to late-stage AS, demonstrating that 1H NMR-based metabolomics is a reliable tool for early diagnosis and monitoring of the process of AS.

The application of HPLC and microprobe NMR spectroscopy in the identification of metabolites in complex biological matrices.

Nuclear magnetic resonance (NMR)-based metabolomics can be used directly to identify a variety of metabolites in biological fluids and tissues. Metabolite analysis is an important part of life science and metabolomics research. However, the identification of some metabolites using NMR spectroscopy remains a big challenge owing to low abundance or signal overlap. It is important to develop a method to measure these compounds accurately. Two-dimensional NMR spectroscopy, metabolite prediction software packages, and spike-in experiments with authentic standards are often used to solve these problems, but they are costly and time-consuming. In this study, methods were developed to identify metabolites in complex biological mixtures using both high-performance liquid chromatography (HPLC) and off-line microprobe NMR spectroscopy. With use of these methods, 83 and 73 metabolites were identified in Sprague Dawley rat urine and feces, respectively. Among them, 40 and 45 metabolites, respectively, could not be identified with traditional NMR methods. Our research revealed that the combination of HPLC and NMR techniques could significantly improve the accuracy of trace and overlapped metabolite identification, while offering an effective and convenient approach to identify potential biomarkers in complex biological systems.

New triterpenoids from the leaves of Cyclocarya paliurus.

Six new triterpenoids including four new secodammarane triterpenoid glycosides (1-4), an epoxydammarane triterpenoid glycoside (5), and a new secodammarane triterpenoid (6) were isolated from the ethanolic extract of the leaves of Cyclocarya paliurus. The structures of these compounds were elucidated by spectroscopic analysis methods. Compounds 1-6 were evaluated for their inhibitory activities against α-glucosidase, lipase, DPP-IV, and aldose reductase.