Stepwise Targeted Matching Strategy for Comprehensive Profiling of Xanthohumol Metabolites In Vivo and In Vitro Using UHPLC-Q-Exactive Orbitrap Mass Spectrometer.

Xanthohumol (XN), a natural prenylated flavonoid extracted and isolated from the hop plant (Humulus lupulus), possesses diverse pharmacological activities. Although the metabolites of XN have been investigated in the previous study, a comprehensive metabolic profile has been insufficient in vivo or in vitro until now. The current study was aimed at systematically elucidating the metabolic pathways of XN after oral administration to rats. Herein, a UHPLC-Q-Exactive Orbitrap MS was adopted for the potential metabolites detection. A stepwise targeted matching strategy for the overall identification of XN metabolites was proposed. A metabolic net (53 metabolites included) on XN in vivo and in vitro, as well as the metabolic profile investigation, were designed, preferably characterizing XN metabolites in rat plasma, urine, liver, liver microsomes, and feces. On the basis of a stepwise targeted matching strategy, the net showed that major in vivo metabolic pathways of XN in rats include glucuronidation, sulfation, methylation, demethylation, hydrogenation, dehydrogenation, hydroxylation, and so on. The proposed metabolic pathways in this research will provide essential data for further pharmaceutical studies of prenylated flavonoids and lay the foundation for further toxicity and safety studies.

Comprehensive Profiling of Mangiferin Metabolites In Vivo and In Vitro Based on the "Drug Metabolite Clusters" Analytical Strategy.

Mangiferin, a natural flavonoid compound with multiple biological activities (e.g., anti-inflammatory, anti-oxidant, anti-diabetic, and anti-tumor), has gained increased research interest in recent years. Nevertheless, the metabolic processing of mangiferin has not been fully investigated. In this study, a rapid and efficient analytical strategy named "Drug Metabolite Clusters" was applied for comprehensive profiling of mangiferin metabolites in rat plasma, urine, and feces samples in vivo following oral administration and liver microsomes in vitro. First, the biological samples were pretreated with methanol, acetonitrile, and solid phase extraction (SPE) for further liquid chromatography-mass spectrometry (LC-MS) analysis. Second, the raw data were acquired using ultra-high performance liquid chromatography quadrupole exactive orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap HRMS) under the positive and negative full-scan/dd MS2 modes. Third, mangiferin and its basic metabolites (norathyriol, trihydroxyxanthone, and dihydroxyxanthone) were selected as mangiferin metabolite cluster centers by referring to the relevant literature. Subsequently, according to the pyrolysis law of mass spectrometry, literature reports, and reference material comparison, especially the diagnostic product ions (DPIs), the candidate metabolites were accurately preliminarily identified, and mangiferin metabolite clusters based on metabolite cluster center changes were formed. As a result, a total of 67 mangiferin metabolites (mangiferin included) were detected, including 29 in plasma, 48 in urine, 12 in feces, and 6 in liver microsomes. Among them, trihydroxyxanthones were first detected in rat urine samples after oral mangiferin. We found that mangiferin mainly underwent deglucosylation, dehydroxylation, methylation, glucuronidation, sulfation, and other composite reactions in rats. Herein, we have elucidated the metabolites and metabolic pathways of mangiferin in vivo and in vitro, which provided an essential theoretical basis for further pharmacological studies of mangiferin and a comprehensive research method for the identification of drug metabolites.

[Comparison of alkaloids in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Praeparata based on UHPLC-Q-Exactive Orbitrap MS/MS].

UHPLC-Q-Exactive Orbitrap MS/MS was used to systematically analyze and compare the alkaloids in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Praeparata. After the samples were pretreated in the solid-phase extraction cartridges, 0.1% ammonium hydroxide(A)-acetonitrile(B) was used for gradient elution. The LC-MS method for characterization of alkaloids in the three herbal medicines was established in ESI positive ion mode to collect high resolution MS data of reference substances and samples. On the basis of the information of reference substance cracking behavior, retention time, accurate molecular mass, and related literature, a total of 155 alkaloids were identified in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Prae-parata. Specifically, 130, 127, and 92 alkaloids were identified in Aconiti Kusnezoffii Radix, Aconiti Radix, and Aconiti Lateralis Radix Praeparata, respectively. Monoester alkaloids and amino-alcohol alkaloids were dominant in the three herbal medicines, and the alkaloids in Aconiti Kusnezoffii Radix and Aconiti Radix were similar. This paper can provide a reference for elucidating the pharmacological effects and clinical application differences of the three herbal medicines produced from plants of Aconitum.

Comprehensive Study of In vivo and In vitro Metabolites of Cycloastragenol Based on UHPLC-Q-Exactive Orbitrap Mass Spectrometer.

BACKGROUND: Cycloastragenol (CAG) is a sapogenin derived from the main bioactive constituents of Astragali Radix (AR). However, the current research on CAG metabolism in vivo and in vitro is still inadequate, and the metabolite cluster is incomplete due to incomplete analysis strategy. OBJECTIVE: The objective of this study was to screen and identify the metabolic behavior of CAG in vivo and in vitro. METHODS: A simple and rapid analysis strategy based on UHPLC-Q-Exactive Orbitrap mass spectrometry combined with data-mining processing technology was developed and used to screen and identify CAG metabolites in rat body fluids and tissues after oral administration. RESULTS: As a result, a total of 82 metabolites were fully or partially characterized based on their accurate mass, characteristic fragment ions, retention times, corresponding Clog P values, and so on. Among the metabolites, 61 were not been reported in previous reports. These metabolites (6 metabolites in vitro and 91 in vivo) were generated through reactions of hydroxylation, glucuronidation, sulfation, hydrogenation, hydroxylation, demethylation, deisopropylation, dehydroxylation, ring cleavage, and carboxyl substitution and their composite reactions, and the hydroxylation might be the main metabolic reaction of CAG. In addition, the characteristic fragmentation pathways of CAG were summarized for the subsequent metabolite identification. CONCLUSION: The current study not only clarifies the metabolite cluster-based and metabolic regularity of CAG in vivo and in vitro, but also provides ideas for metabolism of other saponin compounds.

The Analytical Strategy of "Ion Induction and Deduction Based on Net-Hubs" for the Comprehensive Characterization of Naringenin Metabolites In Vivo and In Vitro Using a UHPLC-Q-Exactive Orbitrap Mass Spectrometer.

Naringenin (5,7,4'-trihydroxyflavanone), belonging to the flavanone subclass, is associated with beneficial effects such as anti-oxidation, anticancer, anti-inflammatory, and anti-diabetic effects. Drug metabolism plays an essential role in drug discovery and clinical safety. However, due to the interference of numerous endogenous substances in metabolic samples, the identification and efficient characterization of drug metabolites are difficult. Here, ultra-high-performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry was used to obtain mass spectral information of plasma (processed by three methods), urine, feces, liver tissue, and liver microsome samples. Moreover, a novel analytical strategy named "ion induction and deduction" was proposed to systematically screen and identify naringenin metabolites in vivo and in vitro. The analysis strategy was accomplished by the establishment of multiple "net-hubs" and the induction and deduction of fragmentation behavior. Finally, 78 naringenin metabolites were detected and identified from samples of rat plasma, urine, feces, liver tissue, and liver microsomes, of which 67 were detected in vivo and 13 were detected in vitro. Naringenin primarily underwent glucuronidation, sulfation, oxidation, methylation, ring fission, and conversion into phenolic acid and their composite reactions. The current study provides significant help in extracting target information from complex samples and sets the foundation for other pharmacology and toxicology research.

Correlation between Intestinal Microflora in Irritable Bowel Syndrome and Severity.

Background: Irritable bowel syndrome (IBS) is a common chronic functional gastrointestinal disease accompanied by changes in intestinal microecology. This study investigated the relationship between gut microbiota and disease severity in patients with irritable bowel syndrome (IBS). Methods: An observational study was performed on 60 IBS patients (study group) and 20 healthy controls admitted to our hospital from January 2013 to December 2014. Fecal samples were taken after admission to measure intestinal flora including Bifidobacterium, Lactobacillus, Enterobacter, and Enterococcus, and patient blood was collected to determine serum D-lactate and diamine oxidase (DAO) levels. The gut microbiota and serum markers of the two groups were analyzed. The correlation of gut microbiota index levels and serum markers with disease severity, as well as the correlation between gut microbiota index levels and serum markers, were analyzed. Results: The levels of intestinal Lactobacillus and Bifidobacterium were lower, while the levels of Enterococcus and Enterobacter and serum D-lactate were higher in the study group than those in the control group. The levels of intestinal Lactobacillus and Bifidobacterium were lower, while the levels of Enterococcus and Enterobacter, serum D-lactate, and DAO were higher in patients with moderate IBS than those in patients with mild IBS. The levels of intestinal Lactobacillus and Bifidobacterium were lower in patients with severe IBS than those with moderate IBS, while the levels of Enterococcus and Enterobacter, serum D-lactate, and DAO were higher in patients with severe IBS. There was a significant negative correlation between the levels of Lactobacillus and Bifidobacterium and disease severity and a significant positive correlation between the levels of Enterococcus and Enterobacter, D-lactate, and DAO and disease severity. There was a significant negative correlation between the levels of Lactobacillus and Bifidobacterium and serum D-lactate and DAO, while there was a significant positive correlation between the levels of Enterococcus and Enterobacter and serum D-lactate and DAO (P < 0.05). Conclusion: Intestinal flora, D-lactate, and DAO were abnormal in IBS patients, and intestinal flora was closely correlated with disease severity, D-lactate, and DAO levels.

Serial five-membered lactone ring ions in the treatment of Alzheimer's diseases-comprehensive profiling of arctigenin metabolites and network analysis.

Arctigenin is a phenylpropanoid dibenzylbutyro lactone lignan compound with multiple biological functions. Previous studies have shown that arctigenin have neuroprotective effects in Alzheimer's disease (AD) models both in vivo and in vitro; however, its metabolism in vivo has not been studied. Most traditional analytical methods only partially characterize drug metabolite prototypes, so there is an urgent need for a research strategy that can fully characterize drug metabolites. In the present study, ions fishing with a serial five-membered lactone ring as a fishhook strategy based on ultrahigh-performance liquid chromatography-Q-Exactive Orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) was utilised to characterise the metabolism of arctigenin, and the establishment of this strategy also solved the challenge of creating a comprehensive metabolic profile of neolignan. Based on the proposed strategy, a total of 105 metabolites were detected and characterised, 76 metabolites of which were found in rats and 49 metabolites in liver microsomes. These metabolites were postulated to be produced through oxidation, reduction, hydrolysis, and complex reactions. Subsequently, network pharmacology was utilized to elucidate the mechanism of arctigenin and its main metabolites against Alzheimer's disease, screening 381 potential targets and 20 major signaling pathways. The study on the comprehensive metabolism of arctigenin provides a holistic metabolic profile, which will help to better understand the mechanism of arctigenin in the treatment of Alzheimer's disease (AD) and also provide a basis for the safe administration of arctigenin.