Evaluation of the anti-atherosclerotic effect for Allium macrostemon Bge. Polysaccharides and structural characterization of its a newly active fructan.

Allium Macrostemon Bge. (AMB) is a well-known homology of herbal medicine and food that has been extensively used for thousands of years to alleviate cardiovascular diseases. It contains a significant amount of polysaccharides, yet limited research exists on whether these polysaccharides are responsible for its cardiovascular protective effects. In this study, the anti-atherosclerosis effect of the crude polysaccharides of AMB (AMBP) was evaluated using ApoE-/- mice fed a high-fat diet, along with ox-LDL-induced Thp-1 foam cells. Subsequently, guided by the inhibitory activity of foam cells formation, a major homogeneous polysaccharide named AMBP80-1a was isolated and purified, yielding 11.1 % from AMB. The molecular weight of AMBP80-1a was determined to be 10.01 kDa. AMBP80-1a was firstly characterized as an agavin-type fructan with main chains consisting of →1)-ß-d-Fruf-(2→ and →1,6)-ß-d-Fruf-(2→ linked to an internal glucose moiety, with →6)-ß-d-Fruf-(2→ and ß-d-Fruf-(2→ serving as side chains. Furthermore, the bio-activity results indicated that AMBP80-1a reduced lipid accumulation and cholesterol contents in ox-LDL-induced Thp-1 foam cell. These findings supported the role of AMBP in alleviating atherosclerosis in vivo/vitro. AMBP80-1a, as the predominant homogeneous polysaccharide in AMB, was expected to be developed as a functional agent to prevent atherosclerosis.

Nasal solitary chemosensory cells govern daily rhythm in mouse model of allergic rhinitis.

BACKGROUND: While the daily rhythm of allergic rhinitis (AR) has long been recognized, the molecular mechanism underlying this phenomenon remains enigmatic. OBJECTIVE: We aimed to investigate the role of circadian clock in AR development and to clarify the mechanism by which the daily rhythm of AR is generated. METHODS: AR was induced in mice with ovalbumin. Toluidine blue staining, liquid chromatography-tandem mass spectrometry analysis, real-time quantitative PCR, and immunoblotting were performed with AR and control mice. RESULTS: Ovalbumin-induced AR is diurnally rhythmic and associated with clock gene disruption in nasal mucosa. In particular, Rev-erbα is generally downregulated and its rhythm retained, but with a near-12-hour phase shift. Furthermore, global knockout of core clock gene Bmal1 or Rev-erbα increases the susceptibility of mice to AR and blunts AR rhythmicity. Importantly, nasal solitary chemosensory cells (SCCs) are rhythmically activated, and inhibition of the SCC pathway leads to attenuated AR and a loss of its rhythm. Moreover, rhythmic activation of SCCs is accounted for by diurnal expression of ChAT (an enzyme responsible for the synthesis of acetylcholine) and temporal generation of the neurotransmitter acetylcholine. Mechanistically, Rev-erbα trans-represses Chat through direct binding to a specific response element, generating a diurnal oscillation in this target gene. CONCLUSION: SCCs, under the control of Rev-erbα, are a driver of AR rhythmicity; targeting SCCs should be considered as a new avenue for AR management.

Five-layer-funnel filtering mode discovers effective components of Chinese medicine formulas: Zhishi-Xiebai-Guizhi decoction as a case study.

BACKGROUND: How to screen and identify the effective components in the complex substance system is one of the core issues in achieving the modernization of traditional Chinese medicine (TCM) formulas. However, it is still challenging to systematically screen out the effective components from the hundreds or thousands of components in a TCM formula. PURPOSE: An innovative five-layer-funnel filtering mode stepwise integrating chemical profile, quantitative analysis, xenobiotic profile, network pharmacology and bioactivity evaluation was successfully presented to discover the effective components and implemented on a case study of Zhishi-Xiebai-Guizhi decoction (ZXG), a well-known TCM formula for coronary heart disease (CHD). METHODS: Initially, the chemical profile of ZXG was systemically characterized. Subsequently, the representative constituents were quantitatively analyzed. In the third step, the multi-component xenobiotics profile of ZXG was systemically delineated, and the prototypes absorbed into the blood were identified and designated as the primary bioavailable components. Next, an integrated network of "bioavailable components-CHD targets-pathways-therapeutic effects" was constructed, and the crucial bioavailable components of ZXG against CHD were screened out. Lastly, the bioactivities of crucial bioavailable components were further evaluated to pinpoint effective components. RESULTS: First of all, the chemical profile of ZXG was systemically characterized with the detection of 201 components. Secondly, 37 representative components were quantified to comprehensively describe its content distribution characteristics. Thirdly, among the quantified components, 24 bioavailable components of ZXG were identified based on the multi-component xenobiotic profile. Fourthly, an integrated network led to the identification of 11 crucial bioavailable components against CHD. Ultimately, 9 components (honokiol, magnolol, naringenin, magnoflorine, hesperidin, hesperetin, naringin, neohesperidin and narirutin) exhibiting myocardial protection in vitro were identified as effective components of ZXG for the first time. CONCLUSION: Overall, this innovative strategy successfully identified the effective components of ZXG for the first time. It could not only significantly contribute to elucidating the therapeutic mechanism of ZXG in the treatment of CHD, but also serve as a helpful reference for the systematic discovery of effective components as well as ideal quality markers in the quality assessment of TCM formulas.

Structural and in vivo-in vitro myocardial injury protection features of two novel polysaccharides from Allium macrostemon Bunge and Allium chinense G. Don.

This study aimed to investigate the structural characteristics, in vivo antiatherosclerosis activity, and in vitro myocardial injury protection effects of polysaccharides from Allium macrostemon Bunge and Allium chinense G. Don. Thus, crude polysaccharides of Allium macrostemon Bunge and Allium chinense G. Don significantly reduced serum lipid levels, improved cardiac myocyte morphology and arrangement, and relieved the development of myocardial fibrosis. Meanwhile, the lesion areas of the aorta and aortic valve had evident visual improvements. Furthermore, two main novel purified polysaccharides, namely, AMB-1 and ACGD-1, were isolated and characterized from crude Allium macrostemon Bunge and Allium chinense G. Don fractions, respectively. The purified polysaccharides mainly consisted of fructose and glucose and had molecular weights of 25.22 and 19.53 kDa, respectively. In addition, Fourier transform infrared spectroscopy, methylation, and nuclear magnetic resonance data revealed the primary structures of the AMB1 (or ACGD1) backbone with branched side chains. Scanning electron microscope analysis showed that the purified polysaccharides were both piled together in a lamellar or clastic form with a smooth surface along with linear or irregular bulges. Moreover, the purified polysaccharides both showed nontoxicity on H9c2 cells and effectively dropped hypoxia/reoxygenation-induced apoptosis by the BCL-2/BAX pathway. Overall, the characterization of the structural properties and in vivo and in vitro myocardial injury protection effects of Allium macrostemon Bunge and Allium chinense G. Don polysaccharides enriched our understanding of their nutritional and medicinal values. To the best of our knowledge, this is the first study on the structural characteristics and bioactivities of Allium chinense G. Don polysaccharides.

Deciphering the diurnal rhythm regulating mechanism of flavin-containing monooxygenase 3 in mouse liver.

Circadian genes play an important role in the field of drug metabolism. Flavin-containing monooxygenase 3 is a well-known phase I enzyme which participates in metabolism of many exogenous and endogenous substances, especially production of trimethylamine N-oxide. Here, we aimed to decipher diurnal rhythms of flavin-containing monooxygenase 3 expression and activity, and explore the regulation mechanism by clock genes. Our results showed that its mRNA and protein exhibited robust diurnal rhythms in mouse liver and cell lines. Consistently, significant alterations were observed for in vitro microsomal N-oxidation rates of procainamide, which kept in line with its protein expression at different time in wild-type and reverse erythroblastosis virus α knockout mice. Further, flavin-containing monooxygenase 3 was negatively regulated by E4 promoter-binding protein 4 in AML12 and Hepa1-6 cells, while it was positively influenced by reverse erythroblastosis virus α and brain and muscle ARNT-like protein-1. Moreover, luciferase reporter assays and electrophoretic mobility shift assays showed E4 promoter-binding protein 4 inhibited the transcription of flavin-containing monooxygenase 3 by binding to a D-box1 element (-1606/-1594 bp), while brain and muscle ARNT-like protein-1 positively activated the transcription via direct binding to three E-boxes (-863/-858 bp, -507/-498 bp, and -115/-104 bp) in this enzyme promoter. Taken together, this study would be helpful to reveal the mechanism of clock-controlled drug metabolism and facilitate the practice of chrono-therapeutics.

Single injection by LC-ESI-MS/MS for simultaneous determination of organophosphate tri- and di-esters in plant tissue based on ultrasonic-assisted sequential extraction and single-step purification.

A novel methodology based on ultrasonic-assisted sequential extraction, dispersive-SPE purification, and single-injection on liquid chromatography-tandem mass spectrometry (LC-MS/MS) is proposed, for the first time, to simultaneously measure 14 tri-OPEs and 9 di-OPEs in plant tissues. The samples were successively ultrasonicated with a mixture of hexane:dichloromethane (1:1, v/v) and 8% acetic acid in acetonitrile for extracting tri- and di-OPEs purified with graphitized carbon black and quantitated on LC-MS/MS at the same time. The recoveries of targeted tri- and di-OPEs in the matrix spike ranged from 66% to 120% and 71% to 110% respectively. The proposed method was validated by processing eight types of common vegetables including spinach (Spinacia oleracea L.), lettuce (Lactuca sativa), carrot (Daucus carota var. sativa Hoffm.), sweet potato (Solanum tuberosum L.), cucumber (Cucumis sativus L.), tomato (Solanum lycopersicum L.), green beans (Phaseolus vulgaris), and cowpeas (Vigna unguiculata), with the recoveries of surrogates ranging from 84% to 98%.

A compounds annotation strategy using targeted molecular networking for offline two-dimensional liquid chromatography-mass spectrometry analysis: Yupingfeng as a case study.

Component overlapping and long-time consumption hinder the data processing of offline two-dimensional liquid chromatography mass spectrometry (offline 2D-LC MS) system. Although molecular networking has been commonly employed in data processing of liquid chromatography mass spectrometry (LC-MS), its application in offline 2D-LC MS is challenged by voluminous and redundant data. In light of this, for the first time, a data deduplication and visualization strategy combining hand-in-hand alignment with targeted molecular networking (TMN) for compounds annotation of offline 2D-LC MS data was developed and applied to the chemical profile of Yupingfeng (YPF), a classical traditional Chinese medicine (TCM) prescription, as a case study. Firstly, an offline 2D-LC MS system was constructed for the separation and data acquisition of YPF extract. Then the data of 12 fractions derived from YPF were deconvoluted and aligned as a whole data file by hand-in-hand alignment, resulting in a 49.2% reduction in component overlapping (from 17951 to 9112 ions) and an improvement in the MS2 spectrum quality of precursor ions. Subsequently, the MS2-similarity adjacency matrix of focused parent ions was computed by a self-building Python script, which realized the construction of an innovative TMN. Interestingly, the TMN was found to be able to efficiently distinguish and visualize the co-elution, in-source fragmentations and multi-type adduct ions in a clustering network. Consequently, a total of 497 compounds were successfully identified depending on only seven TMN analysis guided by product ions filtering (PIF) and neutral loss filtering (NLF) for the targeted compounds in YPF. This integrated strategy improved the efficiency of targeted compound discovery in offline 2D-LC MS data, also shown a huge scalability in accurate compound annotation of complex samples. In conclusion, our study developed available concepts and tools while providing a research paradigm for efficient and rapid compound annotation in complex samples such as TCM prescriptions, with YPF as an example.

Analysis and subcellular distribution of organophosphate esters (OPEs) in rice tissues.

Recent studies have identified the ability of plants to uptake and translocate organophosphate esters (OPEs) within cells. In response to the increasing interest in OPEs and their occurrence in paddy fields and rice, the current study aimed to present an effective and sensitive GC-MS based methodology for quantitative determination of 11 OPEs with octanol-water coefficients ranging from 1.6 to 10. Rice was sonicated with hexane and dichloromethane, and fractionated on two columns: one consisting of neutral alumina, and neutral silica, and the other consisting of graphitized carbon black. Method precision was validated using spiked rice (n = 30) and procedural blanks (n = 9). The mean recovery of matrix spikes for all target OPEs were within 78-110% with relative standard deviation lower than 25%, with a few exceptions. This method was applied to process the wild rice (O. sativa) in which tri-n-propyl phosphate was the dominant targeted OPE. The recoveries of surrogate standards were 81 ± 17% for d12- tris(2-chloroethyl) phosphate and 95 ± 8.8% for 13C12- triphenyl phosphate. The developed method was further used to examine the recoveries of target OPEs in the subcellular structure of rice tissues, including cell wall, cell organelles, cell water-soluble fractions, and cell residue. Recoveries of most target OPEs were in the range of 50-150%; however, ion enhancement was observed for four OPEs in root and shoot tissues. Hydrophobic OPEs accumulated in the cell wall, cell residue, and cell organelles while chlorinated OPEs mainly distributed in the cell water-soluble fraction. These results provide new insight for the ecological risk assessment of OPEs in an important food staple.

Pharmacokinetics integrated with network pharmacology to clarify effective components and mechanism of Wendan decoction for the intervention of coronary heart disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Coronary heart disease (CHD), one of the leading causes of mortality in the world among chronic non-infectious diseases, is closely associated with atherosclerosis, which ultimately leads to myocardial injury. Wendan decoction (WDD), a classical famous formula, exerted an intervention effect on CHD according to numerous reports. However, the effective components and underlying mechanisms for the treatment of CHD have not been fully elucidated. AIM OF THE STUDY: An in-depth investigation of the effective components and mechanisms of WDD for the intervention of CHD was further explored. MATERIALS AND METHODS: Firstly, based on our previous metabolic profile results, a quantification method for absorbed components was established by ultra-performance liquid chromatography triple quadrupole-mass spectrometry (UPLC-TQ-MS) and applied to the pharmacokinetics study of WDD. Then the network pharmacology analysis for considerable exposure components in rat plasma was employed to screen key components of WDD. Gene ontology and KEGG pathway enrichment analysis were further performed to obtain putative action pathways. The effective components and mechanism of WDD were confirmed by in vitro experiments. RESULTS: A rapid and sensitive quantification method was successfully applied to the pharmacokinetic study of 16 high-exposure components of WDD at three different doses. A total of 235 putative CHD targets were obtained for these 16 components. Then, 44 core targets and 10 key components with high degree values were successively screened out by the investigation of protein-protein interaction and the network of "herbal medicine-key components-core targets". Enrichment analysis suggested that the PI3K-Akt signaling pathway was closely related to this formula's therapeutic mechanism. Furthermore, pharmacological experiments demonstrated that 5 of 10 key components (liquiritigenin, narigenin, hesperetin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and isoliquiritigenin) significantly enhanced DOX-induced H9c2 cell viability. The cardioprotective effects of WDD against DOX-induced cell death through the PI3K-Akt signaling pathway were verified by western blot experiments. CONCLUSION: The integration of pharmacokinetics and network pharmacology approaches successfully clarified 5 effective components and therapeutic mechanism of WDD for the intervention of CHD.

Reprogramming of rhythmic liver metabolism by intestinal clock.

BACKGROUND & AIMS: Temporal oscillations in intestinal nutrient processing and absorption are coordinated by the local clock, which leads to the hypothesis that the intestinal clock has major impacts on shaping peripheral rhythms via diurnal nutritional signals. Here, we investigate the role of the intestinal clock in controlling liver rhythmicity and metabolism. METHODS: Transcriptomic analysis, metabolomics, metabolic assays, histology, quantitative (q)PCR, and immunoblotting were performed with Bmal1-intestine-specific knockout (iKO), Rev-erba-iKO, and control mice. RESULTS: Bmal1 iKO caused large-scale reprogramming of the rhythmic transcriptome of mouse liver with a limited effect on its clock. In the absence of intestinal Bmal1, the liver clock was resistant to entrainment by inverted feeding and a high-fat diet. Importantly, Bmal1 iKO remodelled diurnal hepatic metabolism by shifting to gluconeogenesis from lipogenesis during the dark phase, leading to elevated glucose production (hyperglycaemia) and insulin insensitivity. Conversely, Rev-erba iKO caused a diversion to lipogenesis from gluconeogenesis during the light phase, resulting in enhanced lipogenesis and an increased susceptibility to alcohol-related liver injury. These temporal diversions were attributed to disruption of hepatic SREBP-1c rhythmicity, which was maintained via gut-derived polyunsaturated fatty acids produced by intestinal FADS1/2 under the control of a local clock. CONCLUSIONS: Our findings establish a pivotal role for the intestinal clock in dictating liver rhythmicity and diurnal metabolism, and suggest targeting intestinal rhythms as a new avenue for improving metabolic health. IMPACT AND IMPLICATIONS: Our findings establish the centrality of the intestinal clock among peripheral tissue clocks, and associate liver-related pathologies with its malfunction. Clock modifiers in the intestine are shown to modulate liver metabolism with improved metabolic parameters. Such knowledge will help clinicians improve the diagnosis and treatment of metabolic diseases by incorporating intestinal circadian factors.

PER2 integrates circadian disruption and pituitary tumorigenesis.

Rationale: The role of circadian clock in pituitary tumorigenesis remains elusive. Here we investigate whether and how circadian clock modulates the development of pituitary adenomas. Methods and Results: We found altered expression of pituitary clock genes in patients with pituitary adenomas. In particular, PER2 is prominently upregulated. Further, jetlagged mice with PER2 upregulation have accelerated growth of GH3 xenograft tumor. Conversely, loss of Per2 protects mice against developing estrogen-induced pituitary adenoma. Similar antitumor effect is observed for SR8278, a chemical that can decrease pituitary PER2 expression. RNA-seq analysis suggests involvement of cell cycle disturbance in PER2 regulation of pituitary adenoma. Subsequent in vivo and cell-based experiments validate that PER2 induces pituitary expression of Ccnb2, Cdc20 and Espl1 (three cell cycle genes) to facilitate cell cycle progression and inhibit apoptosis, thereby promoting pituitary tumorigenesis. Mechanistically, PER2 regulates the transcription of Ccnb2, Cdc20 and Espl1 through enhancing the transcriptional activity of HIF-1α. HIF-1α trans-activates Ccnb2, Cdc20 and Espl1 via direct binding to its specific response element in the gene promoters. Conclusion: PER2 integrates circadian disruption and pituitary tumorigenesis. These findings advance our understanding of crosstalk between circadian clock and pituitary adenomas and highlight the relevance of clock-based approaches in disease management.

Determination of polyoxymethylene-water partition coefficients for diverse organophosphate esters (OPEs) and prediction of the free-dissolved OPEs in OPE-contaminated soil.

Increasing attention on the estimation of bioavailability of organophosphate esters (OPEs) in soil or sediment has urged the development of techniques to measure soil-/sediment-associated porewater concentrations of OPEs. In this study, we investigated the sorption dynamics of 8 OPEs to polyoxymethylene (POM) spanning one order of magnitude of aqueous OPE concentrations and proposed POM-water partitioning coefficients (Kpom/w) for OPEs. The results showed that the Kpom/w values were mainly affected by the hydrophobicity of OPEs. OPEs with high solubility preferentially partitioned into the aqueous phase indicated by the low log Kpom/w values; while lipophilic OPEs were observed to be taken up by POM. The concentration of lipophilic OPEs in the aqueous phase had a strong impact on their sorption dynamics on POM, with higher aqueous concentrations accelerating the sorption dynamics and shortening the time for equilibration. We proposed that the required time to reach equilibration for targeted OPEs should be 42 d. The proposed equilibration time and Kpom/w values were further validated by applying POM to soil artificially contaminated with OPEs to measure OPEs soil-water partitioning coefficients (Ks). The variations of Ks among soil types implied the need to elucidate the effects of soil properties and chemical properties of OPEs on their distribution between soil and water in the future.

Cytochrome P450 metabolism studies of [6]-gingerol, [8]-gingerol, and [10]-gingerol by liver microsomes of humans and different species combined with expressed CYP enzymes.

Gingerols, mainly [6]-gingerol (6G), [8]-gingerol (8G), and [10]-gingerol (10G), are the functional and specific pungent phytochemicals in ginger. However, poor oral bioavailability limits their applications owing to extensive metabolism. The present study aims to characterize the cytochrome P450 (CYP) metabolic characteristics of 6G, 8G, and 10G by using pooled human liver microsomes (HLM), different animal liver microsomes, and the expressed CYP enzymes. It is shown that NADPH-dependent oxidation and hydrogenation metabolisms of gingerols are the main metabolic types in HLM. With the increase of the carbon chain, the polarity of gingerols decreases and the formation of hydrogenated metabolites is more efficient (CLint: 1.41 µL min-1 mg-1 for 6G, 7.79 µL min-1 mg-1 for 8G and 14.11 µL min-1 mg-1 for 10G), indicating that the phase I metabolism of gingerols by HLM varied with the chemical structure of the substrate. The phase I metabolism of gingerols revealed considerable species variations, and compared to HLM, novel metabolites such as (3S,5S)-gingerdiols and demethylated metabolites are generated in some animal liver microsomes. The primary enzymes involved in the oxidized and demethylated metabolism of these gingerols are CYP1A2 and CYP2C19, but their affinities for gingerols are not the same. CYP2D6 and CYP2B6 contributed significantly to the formation of (3R,5S)-[8]-gingerdiol and (3R,5S)-[10]-gingerdiol, respectively; however, the enzyme responsible for the production of (3R,5S)-[6]-gingerediol is yet to be identified. Some metabolites in microsomes cannot be detected by the 12 investigated CYP enzymes, which may be related to the combined effects of multiple enzymes in microsomes, the different affinity of mixed liver microsomes and CYP enzymes, gene polymorphisms, etc. Overall, this work provides a deeper knowledge of the influence of CYP metabolism on the gingerols, as well as the mode of action and the possibility for drug-herbal interactions.

Characterization of volatile organic compounds with anti-atherosclerosis effects in Allium macrostemon Bge. and Allium chinense G. Don by head space solid phase microextraction coupled with gas chromatography tandem mass spectrometry.

Introduction: Allium macrostemon Bge. (AMB) and Allium chinense G. Don (ACGD) are both edible Allium vegetables and named officinal Xiebai (or Allii Macrostemonis Bulbus) in East Asia. Their medicinal qualities involve in lipid lowering and anti-atherosclerosis effects. And steroidal saponins, nitrogenous compounds and sulfur compounds are like the beneficial components responsible for medicinal functions. Sulfur compounds are the recognized main components both in the volatile oils of AMB and ACGD. Besides, few researches were reported about their holistic chemical profiles of volatile organic compounds (VOCs) and pharmacodynamic effects. Methods: In this study, we first investigated the lipid-lowering and anti-atherosclerotic effects of volatile oils derived from AMB and ACGD in ApoE -/- mice with high fat and high cholesterol diets. Results: The results showed the volatile oils of AMB and ACGD both could markedly reduce serum levels of TG, TC, and LDL-C (p < 0.05), and had no alterations of HDL-C, ALT, and AST levels (p > 0.05). Pathological results displayed they both could obviously improve the morphology of cardiomyocytes and the degree of myocardial fibrosis in model mice. Meanwhile, oil red O staining results also proved they could apparently decrease the lesion areas of plaques in the aortic intima (p < 0.05). Furthermore, head space solid phase microextraction coupled with gas chromatography tandem mass spectrometry combined with metabolomics analysis was performed to characterize the VOCs profiles of AMB and ACGD, and screen their differential VOCs. A total of 121 and 115 VOCs were identified or tentatively characterized in the volatile oils of AMB and ACGD, respectively. Relative-quantification results also confirmed sulfur compounds, aldehydes, and heterocyclic compounds accounted for about 85.6% in AMB bulbs, while approximately 86.6% in ACGD bulbs were attributed to sulfur compounds, ketones, and heterocyclic compounds. Multivariate statistical analysis showed 62 differentially expressed VOCs were observed between AMB and ACGD, of which 17 sulfur compounds were found to be closely associated with the garlic flavor and efficacy. Discussion: Taken together, this study was the first analysis of holistic chemical profiles and anti-atherosclerosis effects of AMB and ACGD volatile oils, and would benefit the understanding of effective components in AMB and ACGD.

Inhibition of estrogen sulfation by Xian-Ling-Gu-Bao capsule.

Xian-Ling-Gu-Bao capsule (XLGB) is a widely prescribed traditional Chinese medicine used for the treatment of osteoporosis. However, it significantly elevates levels of serum estrogens. Here we aimed to assess the dominant contributors of sulfotransferase (SULT) enzymes to the sulfation of estrogens and identify the effective inhibitors of this pathway in XLGB. First, estrone, 17ß-estradiol, and estriol underwent sulfation in human liver S9 extracts. Phenotyping reactions and enzyme kinetics assays revealed that SULT1A1, 1A2, 1A3, 1C4, 1E1, and 2A1 all participated in estrogen sulfation, with SULT1E1 and 1A1 as the most important contributors. The incubation system for these two active enzymes were optimized with Tris-HCl buffer, DL-Dithiothreitol (DTT), MgCl2, adenosine 3'-phosphate 5'-phosphosulfate (PAPS), protein concentration, and incubation time. Then, 29 compounds in XLGB were selected to investigate their inhibitory effects and mechanisms against SULT1E1 and 1A1 through kinetic modelling. Moreover, in silico molecular docking was used to validate the obtained results. And finally, the prenylated flavonoids (isobavachin, neobavaisoflavone, etc.) from Psoralea corylifolia L., prenylated flavanols (icariside II) from Epimedium brevicornu Maxim., tanshinones (dihydrotanshinone, tanshinone II-A,) from Salvia miltiorrhiza Bge., and others (corylifol A, corylin) were identified as the most potent inhibitors of estrogen sulfation. Taken together, these findings provide insights into the understanding regioselectivity of estrogen sulfation and identify the effective components of XLGB responsible for the promotion of estrogen levels.

Metabolic profile and potential mechanisms of Wendan decoction on coronary heart disease by ultra-high-performance quadrupole time of flight-mass spectrometry combined with network pharmacology analysis.

Wendan decoction, a well-known classical traditional Chinese medicine prescription, has been widely used in the clinical application of coronary heart disease for thousands of years. However, due to a lack of research on the overall metabolism of Wendan decoction, the bioavailable components responsible for the therapeutic effects remain unclear, hindering the revelation of its mechanisms against coronary heart disease. Consequently, an efficient joint research pattern combined with characterization of the metabolic profile and network pharmacology analysis was proposed. As a result, a total of 172 Wendan decoction-related xenobiotics (57 prototypes and 115 metabolites) were detected based on the exploration of the typical metabolic pathways of representative pure compounds in vivo, describing their multi-component metabolic characteristics comprehensively. Subsequently, an integrated network of "herbs-bioavailable compounds-coronary heart disease targets-pathways-therapeutic effects" was constructed, and its seven compounds were finally screened out as the key components acting on five main targets of coronary heart disease. Overall, this work not only provided a crucial biological foundation for interpreting the effective components and action mechanisms of Wendan decoction on coronary heart disease but also showed a reference value for revealing the bioactive components of traditional Chinese medicine prescriptions.

Development of a three-step-based novel strategy integrating DMPK with network pharmacology and bioactivity evaluation for the discovery of Q-markers of traditional Chinese medicine prescriptions: Danlou tablet as an example.

BACKGROUND: Quality marker (Q-marker) serves an important role in promoting the standardization of the quality of traditional Chinese medicine (TCM) prescriptions. However, discovering comprehensive and representative Q-markers from TCM prescriptions composed of multiple components remains difficult. PURPOSE: A three-step-based novel strategy integrating drug metabolism and pharmacokinetics (DMPK) with network pharmacology and bioactivity evaluation was proposed to discover the Q-markers and applied to a research example of Danlou tablet (DLT), a famous TCM prescription with remarkable and reliable clinical effects for coronary heart disease (CHD). METHODS: Firstly, the metabolic profile in vivo of DLT was systemically characterized, and the pharmacokinetic (PK) properties of PK markers were then investigated. Secondly, an integrated network of "PK markers - CHD targets - pathways - therapeutic effects" was established to screen out the crucial PK markers of DLT against CHD. Thirdly, the crucial PK markers that could exhibit strong myocardial protection activity in the H9c2 cardiomyocyte model were selected as the candidate Q-markers of DLT. According to the proportion of their Cmax value in vivo, the candidate Q-markers were configured into a composition; the bioactivity was then evaluated to confirm their synergistic effect and justify their usage as Q-markers. RESULTS: First of all, a total of 110 DLT-related xenobiotics (35 prototypes and 75 metabolites) were detected in bio-samples, and the pharmacokinetic properties of 13 PK markers of DLT were successfully characterized, revealing the quality transitivity and traceability from prescription to in vivo. Then, 6 crucial PK markers with three topological features (degree, betweenness, and closeness) greater than the average values in the pharmacology network were screened out as the key components of DLT against CHD. Furthermore, among these 6 crucial PK markers, 5 components (puerarin, alisol A, daidzein, paeoniflorin, and tanshinone IIA) with strong myocardial protection activity were chosen as the candidate Q-markers to constitute a new composition. The composition activated the expression of the PI3K/AKT pathway and exhibited strong myocardial protection activity, and the effective concentrations (nM level) of these components in the composition were significantly lower than their individually effective concentrations (µM level), indicating that there was a certain synergistic effect between them. Hence, the 5 components with multiple properties, including testability, quality transitivity and traceability from prescription to in vivo, effectiveness, and compatibility contribution, were defined as comprehensive and representative Q-markers of DLT. CONCLUSION: This study not only presented a novel idea for the revelation of comprehensive and representative Q-markers in quality control research of TCM prescriptions, but also identified the reasonable Q-markers of DLT for the first time to improve the quality control level of DLT.

Identification of naturally occurring inhibitors in Xian-Ling-Gu-Bao capsule against the glucuronidation of estrogens.

Xian-Ling-Gu-Bao (XLGB) capsule, a well-known traditional Chinese medicine prescription, is widely used for the treatment of osteoporosis. It could significantly increase the levels of estrogen in ovariectomized rats and mice. However, this working mechanism has not been well elucidated. Considering that UDP-glucuronosyltransferase (UGT) enzymes are the important enzymes that inactivate and regulate estrogen activity in vivo, this study aimed to identify the bioactive compounds from XLGB against the glucuronidation of estrogens. First, thirty compounds were considered as candidate bioactive compounds based on our previous studies including pharmacological evaluation, chemical profiles, and metabolic profiles. Second, the characteristics of estrogen glucuronidation by uridine diphosphate glucuronic acid (UDPGA)-supplemented human liver microsomes (HLM), human intestine microsomes (HIM), and expressed UGT enzymes were determined, and the incubation systems of their key UGT enzymes were optimized. Then, inhibitory effects and mechanisms of XLGB and its main compounds toward the key UGT isozymes were further investigated. As a result, estrogen underwent efficient glucuronidation by HLM and HIM. UGT1A10, 1A1, and 2B7 were mainly responsible for the glucuronidation of estrone, ß-estradiol, and estriol, respectively. For E1 and E2, UGT1A10 and 1A1 tended to mediate estrogen-3-O-glucuronidation, while UGT2B7 preferred catalyzing estrogen-16-O-glucuronidation. Furthermore, the incubation system for active UGT isoforms was optimized including Tris-HCl buffer, detergents, MgCl2 concentration, ß-glucuronidase inhibitors, UDPGA concentration, protein concentration, and incubation time. Based on optimal incubation conditions, eleven, nine, and nine compounds were identified as the potent inhibitors for UGT1A10, 1A1, and 2B7, respectively (IC50 < 4.97 µM and Ki < 3.35 µM). Among them, six compounds (bavachin, isobavachin, isobavachalcone, neobavaisoflavone, corylifol A, and icariside II) simultaneously demonstrated potent inhibitory effects against these three active enzymes. Prenylated flavanols from Epimedium brevicornu Maxim., prenylated flavonoids from Psoralea corylifolia L., and salvianolic acids from Salvia miltiorrhiza Bge. were characterized as the most important and effective compounds. The identification of potent natural inhibitors of XLGB against the glucuronidation of estrogen laid an important foundation for the pharmacodynamic material basis.

Dose Optimisation of Posaconazole and Therapeutic Drug Monitoring in Pediatric Patients.

Experience in the clinical use of posaconazole (PCZ) in pediatric patients is limited, and no specific dose recommendations exist. This study aimed to investigate an appropriate dosing regimen, and assess the exposure-response relationships of PCZ in children. We reviewed the medical records of inpatients aged <18 years who subjected to PCZ concentrations monitoring. Clinical data, PCZ dosing and monitoring data were collected. A total of 375 PCZ trough concentrations (C min) from 105 pediatric patients were included. For children receiving PCZ for prophylaxis, the median doses required to achieve the therapeutic range at the ages of <6, 6-12 and >12 years were 14.80, 14.52 and 12.90 mg/kg/day, respectively (p = 0.001); and for those receiving PCZ for treatment, the median doses were 23.50, 20.96 and 15.38 mg/kg/day, respectively (p = 0.001). Among children taking PCZ for prophylaxis, 12% developed a proven or probable breakthrough IFIs; the median PCZ concentrations were significantly lower than those children with successful treatment response (0.43 versus 1.20 µg mL-1; p < 0.001). 79.2% patients taking PCZ for treatment had a positive clinical response, and the median PCZ concentrations were significantly higher than those children with disease progression (1.06 versus 0.53 µg mL-1; p = 0.024). No association between C min values and hepatotoxicity was observed. Factors such as age, CRP, ALT and co-administration with proton pump inhibitors exhibited significant effects on PCZ C min. It is necessary to adjust the dosing regimens based on PCZ C min to individualize antifungal therapy and provide guidelines for dose adjustment in children.

Population Pharmacokinetics of Mycophenolic Acid in Renal Transplant Patients: A Comparison of the Early and Stable Posttransplant Stages.

Mycophenolic acid (MPA) is an antimetabolic immunosuppressive drug widely used in solid organ transplantation and autoimmune diseases. Pharmacokinetics (PK) of MPA demonstrates high inter- and intra-variability. The aim of this study was to compare the population PK properties of MPA in adult renal transplant patients in the early and stable post-transplant stages and to simulate an optimal dosing regimen for patients at different stages. A total of 51 patients in the early post-transplant period (1 week after surgery) and 48 patients in the stable state (5.5-10 years after surgery) were included in the study. In the two-compartment population PK model, CL/F (23.36 L/h vs. 10.25 L/h) and V/F (78.07 vs. 16.24 L) were significantly different between the two stages. The dose-adjusted area under the concentration time curve (AUCss,12h/dose) for patients in the early stage were significantly lower than those for patients in the stable state (40.83 ± 22.26 mg h/L vs. 77.86 ± 21.34 mg h/L; p < 0.001). According to Monte Carlo simulations, patients with 1.0-1.5 g of mycophenolate mofetil twice daily in the early phase and 0.50-0.75 g twice daily in the stable phase had a high probability of achieving an AUCss,12h of 30-60 mg h/L. In addition, limited sampling strategies showed that two 4-point models (C0-C1-C2-C4 and C1-C2-C3-C6) performed well in predicting MPA exposure by both Bayesian estimate and regression equation and could be applied in clinical practice to assist therapeutic drug monitoring of MPA.