Rat Feces Research Articles

Cold exposure, gut microbiota, and hypertension: A mechanistic study

Cold exposure has been recognized as an important risk factor for hypertension, and altered gut microbiota has been reported to be associated with hypertension. We hypothesized that there is a plausible relationship between gut microbiota and cold-induced hypertension (CIH). Therefore, we explored the potential link between the gut microbiota and its metabolites with CIH. Male Sprague-Dawley (SD) rats were randomly divided into the normal temperature group (NT, 20 ± 2 °C) and the cold exposure group (CE, 4 ± 1 °C), and faecal bacteria cross-transplantation was performed after six weeks. We analyzed the gut microbiota of rats using the 16S rDNA sequence and measured the blood pressure of rats and the content of short-chain fatty acids in rat faeces. After six weeks of cold exposure, the CIH rat model was successfully established. The cold exposure reduced the diversity of the gut microbiota, increased the abundance of potentially pathogenic and conditionally pathogenic bacteria (e.g., Quinella, Rothia, and Senegalimassilia genera), and reduced the abundance of beneficial bacteria (e.g., Lactobacillus genus) and butyric acid-producing bacteria (e.g., Lachnospiraceae UCG-008 and Ruminococcaceae UCG-013 genera). Faecal bacteria cross-transplantation altered gut microbiota composition and regulated blood pressure levels. The NT group rats transplanted with CIH rats' faecal bacteria were enriched with certain conditional pathogenic bacteria such as Prevotellaceae UCG-003 genus. The CIH rats transplanted with faecal bacteria from the NT group rats were enriched with beneficial bacteria such as Bacteroides genus. In addition, we found a significant reduction in butyric acid levels in CIH rats, which may be related to the increase in blood pressure. In conclusion, CIH is associated with altered gut microbiota and reduced butyric acid. Our findings provide novel insights for the prevention and treatment of CIH by modulating the gut microbiota through supplementation of beneficial bacteria/butyrate.

Metabolite profiling analysis of plasma, urine, and feces of rats after oral administration of Flos Chrysanthemi Indici preparation through UHPLC-Q-Exactive-MS combined with pharmacokinetic study of markers by UHPLC-QQQ-MS/MS.

Zhenju Antihypertensive Tablet is one of the Flos Chrysanthemi Indici preparations (FCIP) with antihypertensive effect. In order to investigate its process in vivo, a method using ultra-high performance liquid chromatography coupled with quadruple Exactive mass spectrometry (UHPLC-Q-Exactive-MS) was established for comprehensive analysis of the absorbed active component and metabolites in rat plasma, urine, and feces after the oral administration of FCIP. As a result, a total of 61 FCIP-related xenobiotics were identified, including 35 in plasma (25 prototypes, 10 metabolites), 40 in urine (23 prototypes, 17 metabolites), and 25 in feces (12 prototypes, 13 metabolites). The metabolism reactions included phase I reactions (such as oxidation, methylation, and reduction) and phase II reactions (such as sulfate conjugation and glucuronide conjugation). Meanwhile, a verified and optimized ultra-performance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method was developed for the simultaneous investigation of the pharmacokinetic profiles of four markers in FCIP. The results showed that all of the four components had good oral absorption effect. This study simultaneously investigated the comprehensive metabolic profiling of FCIP in rat plasma, urine, and feces after the oral administration as well as the four components pharmacokinetic behavior. The results can provide the therapeutic material basis for FCIP.

Pharmacokinetics, Tissue Distribution, Metabolism and Excretion of a Novel COX-2 Inhibitor, Vitacoxib, in Rats.

The objectives of this study were to elucidate absorption, tissue distribution, excretion, and metabolism of vitacoxib, a novel selective cyclooxygenase-2 inhibitor, in Wistar rats. Vitacoxib was detected in most tissues within 15 min, suggesting that it was well distributed. Moreover, it could cross the intestinal barrier. Vitacoxib was mainly eliminated as two metabolites. Nine proposed metabolites of vitacoxib were found in the plasma, bile, urine, and feces of rats. Two main metabolites, 4-(4-chloro-1-(5-(methyl-sulfonyl) pyridin-2-yl)-1H-imidazol-5-yl) phenyl methanol (M1) and 4-(4-chloro-1-(5-(methyl-sulfonyl) pyridin-2-yl)-1H-imidazol-5-yl) benzoic acid (M2), were identified in rat feces and urine. Further, the authentic standards of M1 and M2 were synthesized to confirm their structures. The carboxylic acid derivative was the major metabolite of vitacoxib excreted in the urine and feces. Hydroxylation of the aromatic methyl group of vitacoxib and additional oxidation of the hydroxymethyl metabolite to a carboxylic acid metabolite were the proposed metabolic pathways. Vitacoxib displayed a high AUClast (4895.73 ± 604.34 ng·h/ml), long half-life (4.25 ± 0.30 h), slow absorption (Tmax, 5.00 ± 2.00 h), and wide tissue distribution in rats. Our findings provide significant information for the further development and investigation of vitacoxib as an effective nonsteroidal anti-inflammatory agent, and highly its potential for use future in a clinical setting.

Streptobacillus notomytis Bacteremia after Exposure to Rat Feces

To determine the source of Streptobacillus notomytis bacteremia in a woman in Japan with signs of rat-bite fever, we examined rat feces from her home. After culture and PCR failed to identify the causative organism in the feces, next-generation sequencing detected Streptobacillus spp., illustrating this procedure’s value for identifying causative environmental organisms.

Correlation analysis of intestinal flora and pathological process of type 2 diabetes mellitus

To observe the relationship between the different stages of type 2 diabetes mellitus (T2DM) and the intestinal flora and verify its underlying mechanism. T2DM rats were generated by high-fat diet (HFD) combined with intraperitoneal streptozotocin (STZ) injection. The rats were divided into four groups: the control group (fed with normal feed for 1 month), the HFD group (fed with HFD for 1 month), the T2DM group (HFD combined with STZ and blood glucose ≥11.1 mM), and the unformed T2DM model (Un-mod) group (HFD combined with STZ and blood glucose <11.1 mM). Feces were collected, and bacterial communities in the fecal samples were analyzed by 16S rRNA gene sequencing. The content of short-chain fatty acids (SCFAs) in feces was measured by gas chromatography. Western blot and quantitative real-time polymerase chain reaction were used to detect the expression of G protein-coupled receptor 41 (GPR41) and GPR43. At different stages of T2DM, the intestinal flora and SCFAs content of rats were significantly decreased (all P < .05). Our results indicated that g__Prevotella had a significant negative correlation, and g__Ruminococcus_torques_group and g__lachnoclastic had a significant positive correlation with blood glucose. The content of SCFAs, in particular acetate and butyrate, in rat feces of different stages of T2DM were significantly reduced, as well as GPR41 and GPR43 expression. The results in the Un-mod group were similar to the T2DM group, and the expression of GPR41 and GPR43 proteins were significantly higher than those in the T2DM group (both P < .001). The intestinal flora–SCFAs–GPR41/GPR43 network may be important in the development of T2DM. Decreasing blood glucose levels by regulating the intestinal flora may become a new therapeutic strategy for T2DM, which has very important clinical and social values.

Enhancing the Antibacterial Activity of Quinoa Fermented by Probiotics: In vitro and In vivo Study

Fermentation of quinoa by probiotics provides higher nutritional value and can be considered as a significant source of bioactive compounds and alive probiotics for the human body. Moringa leaves powder (MLP) at the levels of 0.25 and 0.50 % were used as an additional prebiotic source to supply quinoa fermentation by Lactobacillus plantaram ATCC 14917 and Lactobacillus delbrueckii subsp. Bulgaricus EMCC 11102 and produce healthier quinoa products. The results indicated that quinoa products fermented by probiotics showed different antibacterial activity against selected pathogenic bacteria, and supplementation of fermented quinoa products with MLP at bath levels improved its antibacterial activity in most cases. Also, the coliform group counts of rat feces were reduced after feeding for 30 d with fermented quinoa products supplemented with MLP (0.50%). Furthermore, Fermented quinoa products with MLP exhibited acceptable sensory properties compared with fermented quinoa products without MLP. Supplementation of fermented quinoa products with MLP resulted in improving its antibacterial activity with acceptable sensory properties.

Detection and characterization of simvastatin and its metabolites in rat tissues and biological fluids using MALDI high resolution mass spectrometry approach

Simvastatin (SV) is a hypolipidemic agent, and it is the 2nd most widely prescribed lipid-lowering drug. Here, the detection and characterization of SV and its metabolites was studied in selected organs/tissues (lung, liver, brain, heart and kidney) and biological samples (blood, urine and feces) of rats. MALDI Orbitrap MS was used as a high-resolution mass analyzer. 2,5-Dihydroxybenzoic acid (DHB) and 1,5-diaminonaphthalene (DAN) were used as matrices. Several sample loading methods onto the MALDI plate were attempted and dried droplet method was found to be superior. Two different cell disruption methods, pulverization and homogenization, were also evaluated for the optimum sensitivity in MALDI. Pulverization allowed the detection of more metabolites in all organs except the liver, where homogenization led to the detection of more metabolites. Altogether, 13 metabolites were detected, and one metabolite tentatively identified as a reduced product is being reported for the first time. SV and its metabolites were distributed to all the tissues studied except the brain. Overall, the results implied that the pulverized samples were more uniform and larger in surface area, resulting in their more efficient and complete extraction during sample preparation. As shown in the present study, MALDI Orbitrap MS is a useful tool to study drug and metabolite detection and characterization.

Systematic Identification of the Main Constituents from Agrimonia pilosa Ledeb. and Their Metabolites in Rats using HPLC-Q-TOF-MS/MS.

Agrimonia pilosa is a perennial herbaceous flowering plant, commonly known as agrimony or hairy agrimony. The dried aerial parts of this species have been widely used for the treatment of acute diarrhea, hemostasis, and other inflammation-related diseases. However, information on the in vivo metabolism of A.pilosa constituents is limited. In this study, the phytochemical profile of A.pilosa was investigated using HPLC-Q-TOF-MS/MS combined with a nontargeted diagnostic ion network analysis strategy. An information-dependent acquisition method with multiple filters was utilized to screen possible prototypes and metabolites in complex biological matrices. Furthermore, various data-processing techniques were applied to analyze possible prototypes and their metabolites in rat plasma, feces, and urine following oral administration of A.pilosa extract. A total of 62 compounds, which belonged to five main structural classes (21 phenols, 22 flavonoids, 6coumarins, 3 triterpenes, and 10 organic acids), were tentatively identified in A.pilosa. In addition, using our proposed stepwise method, 32 prototypes and 69 metabolites were detected in rat plasma, feces, and urine. The main metabolic pathways after the oral administration of A.pilosa extract were revealed to include methylation, dihydroxylation, demethylation, hydrolysis, sulfation, and glucuronidation. This comprehensive in vivo and in vitro identification of the possible active components in A.pilosa could provide a basis for understanding its various pharmacological activities.

Effects of iodoacetic acid drinking water disinfection byproduct on the gut microbiota and its metabolism in rats

Iodoacetic acid (IAA) is an unregulated disinfection byproduct in drinking water and has been shown to exert cytotoxicity, genotoxicity, tumorigenicity, and reproductive and developmental toxicity. However, the effects of IAA on gut microbiota and its metabolism are still unknown, especially the association between gut microbiota and the metabolism and toxicity of IAA. In this study, female and male Sprague–Dawley rats were exposed to IAA at 0 and 16 mg/kg bw/day daily for 8 weeks by oral gavage. Results of 16S rRNA gene sequencing showed that IAA could alter the diversity, relative abundance and function of gut microbiota in female and male rats. IAA also increased the abundance of genes related to steroid hormone biosynthesis in the gut microbiota of male rats. Moreover, metabolomics profiling revealed that IAA could significantly disturb 6 and 13 metabolites in the feces of female and male rats, respectively. In female rats, the level of androstanediol increased in the IAA treatment group. These results were consistent with our previous findings, where IAA was identified as an androgen disruptor. Additionally, the perturbed gut microbiota and altered metabolites were correlated with each other. The results of this study indicated that IAA could disturb gut microbiota and its metabolism. These changes in gut microbiota and its metabolism were associated with the reproductive and developmental toxicity of IAA.

Optimization and validation of direct gas chromatography-mass spectrometry method for simultaneous quantification of ten short-chain fatty acids in rat feces

Short-chain fatty acids (SCFAs) play key roles in maintaining health and treating disease. Quantification of important fecal SCFAs is necessary to facilitate the clarification of their biological roles. However, the existing quantifying methods mainly depend on complicated precolumn derivatization, and/or are unable to determine formic acid, a SCFA commonly associated with toxicity. In this study, a direct gas chromatography-mass spectrometry (GC-MS) method for simultaneous quantification of ten SCFAs including formic acid in rat feces was developed. The approach was optimized in terms of chromatographic and spectrometric conditions as well as sample preparation. DB-FFAP capillary column with temperature programming was used to get baseline separation and symmetrical peak shape of SCFAs without precolumn derivatization in a relatively short running time (8 min). Multiple reaction monitoring (MRM) scan mode was employed to enhance the sensitivity and selectivity of SCFAs. Acidification with 50% HCl and immediate extraction with diethyl ether were utilized to achieve sample preparation of ten SCFAs from feces. Furthermore, the developed method was validated with wide linear range, high sensitivity and precision, low matrix effect and acceptable accuracy. The established method was successfully applied to compare the contents of fecal SCFAs between normal and immunosuppressed animal models.

Metabonomics combined with 16S rRNA sequencing to elucidate the hypoglycemic effect of dietary fiber from tea residues

Tea residues are rich in dietary fiber, which possesses excellent physicochemical and functional properties in vitro. However, the hypoglycemic effect and mechanism of dietary fiber from tea residues are not clear. The study aimed to investigate the potential hypoglycemic effect of dietary fiber obtained from tea residues fermentation (TRDF) and reveal its related mechanisms of action in terms of both intestinal flora and metabolomics. The type 2 diabetes (T2D) rat model induced by high-fat diet and streptozotocin injection was applied in this study. Four weeks of TRDF intervention could remarkably ameliorate hyperglycemia, severe oxidative stress and insulin resistance of diabetic rats. Additionally, there was a significant increase of short chain fatty acids (SCFAs) concentrations in feces of diabetic rats after TRDF intervention. Furthermore, TRDF played a positive role in relieving intestinal microbiota dysbiosis by enriching beneficial bacteria (S24-7 and Prevotellaceae) and inhibiting harmful bacteria (Desulfovibrionaceae and Clostridiaceae). Metabolomic analysis showed that TRDF improved the amino acid metabolism and citrate cycle. The study elaborated on the hypoglycemic effect and potential mechanisms of TRDF through multiple pathways of gut microbiota and metabolites, which could provide theoretical basis for TRDF as a dietary supplement to manage T2D.

Surface-Enhanced Raman Spectroscopy Analysis of Astragalus Saponins and Identification of Metabolites After Oral Administration in Rats by Ultrahigh-Performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry Analysis.

Astragalus mongholicus Bunge (Fabaceae) is an ancient Chinese herbal medicine, and Astragalus saponins are the main active components, which have a wide range of biological activities, such as immunomodulation, antioxidation, and neuroprotection. In this study, silver nanoparticles obtained by sodium borohydride reduction were used as the enhanced substrate to detect astragaloside I (1), astragaloside II (2), astragaloside III (3), astragaloside IV (4), isoastragaloside I (5), and isoastragaloside II (6) in the phloem, xylem, and cork by surface-enhanced Raman spectroscopy (SERS). In the SERS spectrum of Astragalus slices, the characteristic peaks were observed at 562, 671, 732, 801, 836, 950, 1,026, 1,391, and 1,584 cm−1, among which 950 cm−1 and 1,391 cm−1 were strong SERS signals. Subsequently, the metabolites of the six kinds of Astragalus saponins were identified by UPLC/ESI/Q-TOF-MS. Totally, 80, 89, and 90 metabolites were identified in rat plasma, urine, and feces, respectively. The metabolism of saponins mainly involves dehydration, deacetylation, dihydroxylation, dexylose reaction, deglycosylation, methylation, deacetylation, and glycol dehydration. Ten metabolites (1-M2, 1-M11, 2-M3, 2-M12, 3-M14, 4-M9, 5-M2, 5-M17, 6-M3, and 6-M12) were identified by comparison with reference standards. Interestingly, Astragalus saponins 1, 2, 5, and 6 were deacetylated to form astragaloside IV (4), which has been reported to have good pharmacological neuroprotective, liver protective, anticancer, and antidiabetic effects. Six kinds of active Astragalus saponins from different parts of Astragalus mongholicus were identified by SERS spectroscopy. Six kinds of active Astragalus saponins from different parts of Astragalus mongholicus were identified by SERS spectrum, and the metabolites were characterized by UPLC/ESI/Q-TOF-MS, which not only provided a new method for the identification of traditional Chinese medicine but also provided a theoretical basis for the study of the pharmacodynamic substance basis of Astragalus mongholicus saponins.

Identification and action mechanism of lipid regulating components from Rhei Radix et rhizoma

Ethnopharmacological relevanceRhei Radix et Rhizoma is widely used in Traditional Chinese Medicine to attack stagnation, clear damp heat, relieve fire, cool blood, remove blood stasis and detoxify recorded in Chinese Pharmacopoeia. Modern pharmacological research has showed the extract of Rhei Radix et Rhizoma has the effect of lowering blood lipids, but the main active components and their mechanisms are still not clear. Aim of the studyTo reveal the lipid regulating components from Rhei Radix et Rhizoma and preliminarily explore their related action mechanisms. Materials and methodsA rat model of dyslipidemia was established by administration of a high-fat emulsion via gavage, and the intervention effect of different polar fractions of Rhei Radix et Rhizoma on rat blood lipids as well as their related action mechanisms were preliminarily investigated. The effective components were inferred based on the above tests and identified by high performance liquid chromatography in comparison with reference substances, their UV absorption and high resolution mass spectra characteristics. ResultsThe extract with dichloromethane fraction (DF) containing rhubarb free anthraquinones (aloe-emodin, rhein, emodin, chrysophanol and physcion) significantly regulated the disordered blood lipids, lowered TC and LDLC, reversed TG and increased HDLC level in dyslipidemic rats and also showed lipid-lowering effect on lipid abnormalities in HepG2 cells. DF could alter the signaling pathways such as PPARα and AMPK implicated in lipid metabolism, and it down-regulated the mRNA expression of liver APOA2, SCD-1, HMGCR, SREBP-2 and PCSK9, but up-regulated the expressions of liver APOE, LPL and intestinal ABCG8. Besides, it could change the composition of Firmicutes, Bacteroidetes and Proteobacteria in dyslipidemic rat feces samples. ConclusionsRhubarb free anthraquinones have a significant regulating effect on the levels of serum TC, LDLC and HDLC, and probably possess a bidirectional regulatory effect on TG level in dyslipidemic rats. These effects may be achieved by regulating the expressions of the liver PPARα and SREBP target genes, PCSK9 and the intestinal ABCG8 genes, which are involved in blood cholesterol transport, liver lipid metabolism and intestinal cholesterol excretion. Rhubarb free anthraquinones may also affect energy metabolism by changing the composition of gut microflora related to lipid metabolism in dyslipidemic rats.

In depth search of the Sequence Read Archive database reveals global distribution of the emerging pathogenic fungus Scedosporium aurantiacum.

Scedosporium species are emerging opportunistic fungal pathogens causing various infections mainly in immunocompromised patients, but also in immunocompetent individuals, following traumatic injuries. Clinical manifestations range from local infections, such as subcutaneous mycetoma or bone and joint infections, to pulmonary colonization and severe disseminated diseases. They are commonly found in soil and other environmental sources. To date S. aurantiacum has been reported only from a handful of countries. To identify the worldwide distribution of this species we screened publicly available sequencing data from fungal metabarcoding studies in the Sequence Read Archive (SRA) of The National Centre for Biotechnology Information (NCBI) by multiple BLAST searches. S. aurantiacum was found in 26 countries and two islands, throughout every climatic region. This distribution is like that of other Scedosporium species. Several new environmental sources of S. aurantiacum including human and bovine milk, chicken and canine gut, freshwater, and feces of the giant white-tailed rat (Uromys caudimaculatus) were identified. This study demonstrated that raw sequence data stored in the SRA database can be repurposed using a big data analysis approach to answer biological questions of interest.Lay summaryTo understand the distribution and natural habitat of S. aurantiacum, species-specific DNA sequences were searched in the SRA database. Our large-scale data analysis illustrates that S. aurantiacum is more widely distributed than previously thought and new environmental sources were identified.

Metabolism and Mass Balance in Rats Following Oral Administration of the Novel Antifibrotic Drug Fluorofenidone.

ObjectiveFluorofenidone (AKF-PD) is a novel antifibrotic small-molecule compound. The purpose of this study was to investigate the metabolic and excretory pathways of AKF-PD in rats.MethodsHigh-performance liquid chromatography with mass spectrometric (HPLC-MS) detection was used to analyze the metabolites in rat urine. The metabolites were separated by chromatography and their structure was confirmed. HPLC was used to determine the contents of the parent compound and its metabolites in feces and urine after quantitative administration to study the excretion pathway.ResultsAKF-PD was mainly oxidized to the carboxyl group after methyl hydroxylation. After oral administration, the total amount of the prototype drug and its hydroxylated metabolites and carboxylated metabolites excreted from the urine and feces of rats was 87%. However, most of them are excreted in urine and feces in the form of carboxylated metabolites, and rarely excreted in the form of prototype drugs and hydroxylated metabolites. Which is that the urinary discharge of hydroxylated metabolites, fluorine ketones, and carboxylated metabolites were 0.2%, 1.1%, and 75.2%, respectively, while the fecal discharge were 0.2%, 0.3%, and 10.1%, respectively.ConclusionAKF-PD is mainly oxidized into 2-hydroxymethyl and 5-carboxyl AKF-PD through the Phase I metabolic reaction in rats. AKF-PD is a highly permeable compound classified by biopharmaceutics and is mainly excreted from the urine in the form of metabolites.

UHPLC-Q-TOF-MS/MS-based Metabolite Profiling of Ganpu Tea in Rat Urine and Feces

Ganpu tea is a novel type of beverage produced from Pu-erh tea stuffed in the pericarp of Citrus reticulata “Chachi”. It has gained considerable popularity in China owing to its inviting flavor and health effects. However, the in vivo metabolites of Ganpu tea, which may contribute to its overall health effects, are still unclear. In the present work, rat urine and feces samples were collected after oral administration of Ganpu tea extract (GTE), and then subjected to ultra-high-performance liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS)-based metabolite profiling. As a result, 27 prototype compounds and 41 metabolites derived from caffeic acid, gallic acid, p-coumaric acid, xanthine, catechin, polymethoxyflavone, (PMF) flavanone, and flavone were identified in rat urine and feces. Based on the detected metabolites, the ingested prototype compounds derived from Ganpu tea were found to undergo extensive phase II metabolism in rats, especially and sulfation. These results will be valuable for interpreting the health effects of Ganpu tea.

Effect of Kansui Radix stir-fried with vinegar on fecal metabolomics in Walker-256 rats with ascites

Utilizing metabolomics technology, this study explored the change of fecal endogenous metabolites in Walker-256 rats with malignant ascites after the administration with Kansui Radix(KR) stir-fried with vinegar(VKR), sought the potential biomarkers in feces which were related to the treatment of malignant ascites by VKR and revealed the biological mechanism of water-expelling effect of VKR. Ultra-fast liquid chromatography-quadrupole-time-of-flight mass spectrometry(UFLC-Q-TOF-MS) was employed to detect the feces of rats in all groups. Principle component analysis(PCA) and partial least squares discriminant analysis(PLS-DA) were conducted to achieve pattern recognition. Combining t-test and variable importance in the projection(VIP) enabled the screening of potential biomarkers for the malignant ascites. Metabolic pathway analysis was accomplished with MetaboAnalyst. Correlation analysis was finally conducted integrating the sequencing data of gut microbiota to elucidate the mechanism underlying the water-expelling effect of VKR. The results showed that both KR and VKR could restore the abnormal metabolism of model rats to some extent, with VKR being inferior to KR in the regulation. Eleven potential biomarkers were identified to be correlated with the malignant ascites and five metabolic pathways were then enriched. Four kinds of gut microbiota were significantly related to the potential biomarkers. The water-expelling effect of VKR may be associated with the regulation of phenylalanine metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan, tryptophan metabolism, glycerophospholipid metabolism, and glycosylphosphatidylinositol(GPI)-anchor biosynthesis. This study can provide a scientific basis for comprehensive understandings of the interaction between gut microbiota and host which has relation to the water-expelling effect of VKR and guide the reasonable clinical application of VKR.

Rapid screening and identification of metabolites of murpanicin in rats by UHPLC/Q-TOF-MS/MS combined with diagnostic fragment ions (DFIs) and multiple mass defect filter

Murpanicin is a main coumarin with obvious anti-inflammatory effect isolated from Murraya species. The primary aim of this research is to identify and characterize the possible metabolites of murpanicin in rats. The metabolites generated in the plasma, urine, bile, and feces of rats were identified and characterized employing ultra-high performance liquid chromatography coupled with quadrupole-time of flight tandem mass spectrometry (UHPLC/Q-TOF-MS/MS) based on diagnostic fragment ions (DFIs) and multiple mass defect filter (MMDF). A total of 67 metabolites were identified, among which 55 are phase I and 12 are phase II reacted products. The plausible structures of the metabolites and the probable metabolic pathways were deduced based on the diagnostic fragment ions, mass ppm error, and mass fragmentation pattern, as well as the MetabolitePilot™ software. The majority of phase I metabolites were generated by demethylation, deethylation, dehydrogenation, hydroxylation, and reduction, while phase II metabolites were mainly generated by glucuronidation and sulfation. Moreover, some rare phase II metabolic pathways, such as N-acetylcysteine conjugation, cysteine conjugation, and S-cysteine conjugation were also observed. In conclusion, our study first expounded the metabolites of murpanicin in rats and provided reference for further clarification of the in vivo therapeutic material basis of murpanicin and other 8-prenylcoumarin derivatives. Moreover, UHPLC/Q-TOF-MS combined with MMDF and DFIs has been proved to be an effective method for rapid identification of the homolog-gathered natural products and their metabolites.

Metabolite Identification of Huangqi Jianzhong Tang in Rat Urine and Feces after Oral Administration Based on UHPLC-Q-Exactive-MS.

Huangqi Jianzhong Tang (HQJZ), a famous traditional Chinese medicine formula, is widely used in the treatment of gastrointestinal diseases in China. In this study, an ultra-high-performance liquid chromatography coupled with quadrupole-Exactive mass spectrometry was used to identify the metabolites in rat urine and feces after oral administration of HQJZ coupled with Compound Discover 3.0 software. The possible metabolic pathways were calculated and deduced based on 71 previous detected prototype compounds. As results, 88 compounds were identified in urine and feces, respectively. In urine sample, we identified 20 prototype compounds and 68 related metabolites. Meanwhile, a total of 21 prototype compounds and 67 related metabolites were identified in feces. Among them, flavonoids and saponins were the main ingredients in vivo. Further, we also speculated that HQJZ experienced oxidation, reduction, acetylation, methylation and glucuronic acid reaction in urine and feces. This study established a reliable method for the detection of prototype components and metabolites of traditional Chinese medicines, which would provide helpful information for further research into the active substances of HQJZ.

Pharmacokinetics and metabolism of trans-emodin dianthrones in rats

Ethnopharmacological relevancePolygonum multiflorum Thunb. (PM) is a common traditional Chinese medicine with diverse biological activities of resolving toxins, nourishing livers and promoting hairs. Nevertheless, in recent years hepatotoxic adverse reactions caused by the administration of PM have raised worldwide concerns. In our previous study, we found that emodin dianthrones showed hepatotoxicity and may be potential toxicity markers. However, the metabolic transformation and pharmacokinetic behavior of emodin dianthrones in vivo have still not been elucidated. Aim of the studyTaking trans-emodin dianthrones (TED) as an example, the present study was conducted to investigate the pharmacokinetics and bioavailability of TED in rats and characterized its metabolic transformation in the plasma, urine and feces of rats. Materials and methodsA rapid and sensitive UPLC-qqq-MS/MS method was developed for accurate quantification of TED in plasma and successfully applied to the pharmacokinetic evaluation of TED in rats after intravenous and oral administration. A reliable UFLC-Q-TOF-MS high resolution mass spectrometry combined with a scientific metabolite identification strategy was used to comprehensively characterize the metabolic transformation of TED in plasma, urine and feces in rats. ResultsThe established UPLC-qqq-MS/MS method had a linear range of 1–500 ng/mL, and the method was accurate and reliable to meet the quantitative requirements. When 20 mg/kg TED was given by gavage rats, it was rapidly absorbed into the circulatory system and had a long half-life time of 6.44 h and wide tissue distribution in vivo. While intravenous injection of 0.4 mg/kg TED in rats, it was rapidly metabolized and eliminated with a half-life time of 1.82 h. The oral absorption bioavailability of TED was only 2.83%. Furthermore with a sensitive UFLC-Q-TOF-MS technique and metabolite identification strategy, 21 metabolites were successfully identified, including 11 in plasma, 12 in urine and 18 in feces. The main Ⅰ and Ⅱ phase metabolic processes involved glucuronidation, oxidation, carbonylation, (de)methylation, sulfation and hydrogenation. ConclusionTED could be rapidly absorbed into the blood circulation and widely distributed and slowly metabolized in the body and underwent extensive cleavage and metabolic transformation in vivo. The study provided a basis for in-depth elucidation of the toxicology and mechanism research of TED, but also laid the foundation for further research on the material basis of hepatotoxicity of PM.