Jin-hua Tao

UPLC-Q-TOF/MS-based screening and identification of two major bioactive components and their metabolites in normal and CKD rat plasma, urine and feces after oral administration of Rehmannia glutinosa Libosch extract.

Rehmannia glutinosa is a widely used traditional Chinese medicine (TCM) in clinical practice to tackle chronic kidney disease for thousands of years. However, the in vivo metabolism of its two major bioactive components (catalpol and acteoside) remains unknown. In this paper, a highly sensitive, rapid and robust ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) with MetaboLynx™ software combined with mass defect filtering (MDF) method was established. This validated analysis method was successfully applied to investigate the in vivo metabolic profiles of R. glutinosa extract in normal and chronic kidney disease (CKD) rats. The results showed that a total of 17 metabolites of two parent compounds in normal rats in vivo were tentatively detected and identified according to the characteristics of their protonated ions and relevant literature. While 11 of the metabolites were observed in the CKD rat samples. These metabolites suggested that catalpol was firstly deglycosylated to its aglycone and subsequently to two main metabolites (M1 and M4) by conjugation and hydrogenation respectively and acteoside was mainly metabolized by O-glucuronide conjugation and O-sulphate conjugation. In conclusion, this study showed an insight into the metabolism of R. glutinosa extract in vivo and the proposed metabolic pathways of bioactive components might play a key role in further pharmacokinetic experiments evaluations.

rGO quantum dots/ZnO hybrid nanofibers fabricated using electrospun polymer templates and applications in drug screening involving an intracellular H2O2 sensor

Throughout the years, reported intracellular H2O2 sensors just focused on unrelated measurements of intracellular H2O2 generated from the stimulus of Cd2+, ascorbic acid (AA) etc., leading to difficulty in data interpretation. Here, a novel reduced graphene oxide quantum dots (rGO QDs)/ZnO hybrid nanofibers-based electrochemical biosensor for the detection of intracellular H2O2 released from cancer and normal cells under the stimuli of the corresponding anticancer drugs permits a quantitative study of the interaction between the target drug compound and the cancer cell, which is suitable for candidate drug screening. Nylon 6/6 nanofibers are used as robust templates for the facile fabrication of novel rGO QDs/ZnO hybrid nanofibers via electrospinning followed by a step hydrothermal growth method. The as-made sensor was applied to determine H2O2 released from a prostate cancer cell (PC-3) versus a noncancerous cell (BPH-1) under the stimuli of the corresponding anticancer drugs (apigenin, antisense CK2αetc.). The amount of H2O2 released from the PC-3 cancer cell is about (320 ± 12) amol per cell and about (210 ± 6) amol per cell for the BPH-1 noncancerous cell under the stimuli of specific therapy drug antisense CK2α. These results demonstrate that the rGO QDs/ZnO hybrid nanofibers-based electrochemical biosensor can efficiently detect the distinct amounts of H2O2 released from cancer and noncancer cells.

Novel pleuromutilin derivatives as antibacterial agents: Synthesis, biological evaluation and molecular docking studies

Owing to the increasingly serious problems caused by multidrug resistance in community-acquired infection pathogens, it has become an urgent need to develop new classes of antibiotics for overcoming the resistance. In this paper, we describe the design and synthesis of novel pleuromutilin derivatives containing the (2-aminothiazol-4-yl)-4-methyl group, as well as their in vitro antibacterial activities against Gram-positive clinical bacteria. Most of the tested compounds displayed strong antibacterial activities against these methicillin-susceptible and methicillin-resistant bacteria. Particularly noteworthy compound 15 and its derivative 16e, both showed potent antibacterial properties (0.0625-0.5μg/mL) that are superior to amoxicillin and tiamulin. Molecular docking studies suggested that the amino thiazole ring on the side chains of the pleuromutilin derivatives can in general be accommodated near the mutilin core in the binding pocket, and thus play an important role in the activity of the whole molecule. The findings reported herein may provide a new insight into the design of novel pleuromutilin derivatives for human clinical use.

Biotransformation and metabolic profile of catalpol with human intestinal microflora by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

Traditional Chinese medicine (TCM) has been used in clinical practice for thousands of years. Catalpol, an iridoid glucoside, abundantly found in the root of the common used herb medicine Rehmannia glutinosa Libosch, has been reported to show various biological effects and pharmacological activities. After oral administration, the active ingredient might have interactions with the intestinal bacteria, which could help unravel how the medicine was processed in vivo. In this work, different pure bacteria from healthy human feces were isolated and used to bioconvert catalpol. Ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) technique combined with Metabolynx(™) software was applied to analyze catalpol metabolites. Compared with blank samples, parent compound (M0) and four metabolites (M1-M4) were detected and tentatively identified based on the characteristics of their protonated ions. The metabolites were likely to be: catalpol aglycone (M1), acetylated catalpol (M2), dimethylated and hydroxylated catalpol aglycone (M3), nitrogen-containing catalpol aglycone (M4). M1 and M4 were generated in the majority of the samples like Bacteroides sp. 45. M3 was obtained in several bacterial samples like Enterococcus sp. 8-2 and M2 was detected only in the sample of Enterococcus sp. 43-1. To our knowledge, the metabolic routes and metabolites of catalpol produced by human intestinal bacteria were all firstly reported.

Simultaneous quantification and semi-quantification of ginkgolic acids and their metabolites in rat plasma by UHPLC–LTQ–Orbitrap–MS and its application to pharmacokinetics study

A highly sensitive method using ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS) has been developed and validated for the simultaneous identification and quantification of ginkgolic acids and semi-quantification of their metabolites in rat plasma. For the five selected ginkgolic acids, the method was found to be with good linearities (r>0.9991), good intra- and inter-day precisions (RSD<15%), and good accuracies (RE, from -10.33% to 4.92%) as well. Extraction recoveries, matrix effects and stabilities for rat plasm samples were within the required limits. The validated method was successfully applied to investigate the pharmacokinetics of the five ginkgolic acids in rat plasma after oral administration of 3 dosage groups (900mg/kg, 300mg/kg and 100mg/kg). Meanwhile, six metabolites of GA (15:1) and GA (17:1) were identified by comparison of MS data with reported values. The results of validation in terms of linear ranges, precisions and stabilities were established for semi-quantification of metabolites. The curves of relative changes of these metabolites during the metabolic process were constructed by plotting the peak area ratios of metabolites to salicylic acid (internal standard, IS), respectively. Double peaks were observed in all 3 dose groups. Different type of metabolites and different dosage of each metabolite both resulted in different Tmax.

Simultaneous determination of six short-chain fatty acids in colonic contents of colitis mice after oral administration of polysaccharides from Chrysanthemum morifolium Ramat by gas chromatography with flame ionization detector.

Short-chain fatty acids (SCFAs) produced by the intestinal bacteria are very critical for the intestinal barrier, mucosal cytoprotection and normal intestinal biology. However, accumulation of SCFAs promoted by the polysaccharides from Chrysanthemum morifolium Ramat remains unknown. Thus, it is necessary to investigate SCFAs in the colonic contents of dextran sulfate sodium (DSS) induced colitis mice after oral administration of the polysaccharides from C. morifolium Ramat which is very helpful to unravel how it works. In this study, a rapid and reliable gas chromatographic method with flame ionization detector (GC-FID) for simultaneous determination of six SCFAs such as acetic acid (AA), propionic acid (PA), butyric acid (BA), isobutyric acid (IBA), valeric acid (VA) and isovaleric acid (IVA) has been developed and validated. Under the optimized chromatographic conditions and sample extraction procedure, good separation for 6 target compounds was obtained on a HP-INNOWAX column within 12min. Results revealed that polysaccharides from C. morifolium Ramat positively affected the SCFAs intestinal production. The polysaccharides group had greater SCFAs concentration in colonic content than the DSS-treated group (P<0.05), which was decreased remarkably compared to the normal group (P<0.01). With the decrease of the polysaccharides dosage, the contents of AA, PA and VA increased gradually, while the change of BA concentration was the opposite. There was no significant difference in the content of IBA at the different administration concentrations. And the content of IVA reached the highest concentration 0.953mg/g at lower dose of the polysaccharides. Additionally, oral administration of the polysaccharides prominently attenuated the body weight loss, reduced the disease activity index, rectal bleeding and stool consistency, improved colon shortening and macroscopic score of colitis. Our results indicated that the polysaccharides of C. morifolium Ramat might be used as prebiotic agents to prevent gut dysbiosis and inflammatory bowel disease.

Polysaccharides from Chrysanthemum morifolium Ramat ameliorate colitis rats by modulating the intestinal microbiota community

The gut microflora dysbiosis has been closely related with the inflammatory bowel disease (IBD). In this study, the effect of polysaccharides from Chrysanthemum morifolium Ramat on the gut microbiota was evaluated by ulcerative colitis (UC) rat model. Physiological and pathological analyses suggested that Chrysanthemum polysaccharides possessed notably protective effects on UC in vivo. Based on the Illumina MiSeq platform, 16S rRNA sequencing of the rat colonic contents indicated that the intestinal flora structure remarkably changed in the model rats and the tendency was alleviated to a certain degree by treatment with different dosages of Chrysanthemum polysaccharides. In normal groups, there were more Firmicutes than Bacteroidetes, but this change lost at the pathological state. Following Chrysanthemum polysaccharides, rising Firmicutes/Bacteroidetes ratio was validated. Besides the microbial diversity and the community richness of the UC rats were improved by Chrysanthemum polysaccharides, the composition of intestinal microflora in the model group were also restored after oral administration of Chrysanthemum polysaccharides. The abundance of opportunistic pathogens was decreased (Escherichia, Enterococcus and Prevotella), while the levels of protective bacteria such as Butyricicoccus and Clostridium (butyrate-producing bacteria), Lactobacillus and Bifidobacterium (probiotics), Lachnospiraceae and Rikenellaceae elevated in various degrees. Correlation analysis between intestinal flora and biochemical factors suggested that the relative abundance of protective bacteria was positively correlated with the levels of anti-inflammatory cytokines such as IL-4, IL-10 and IL-11, while aggressive bacteria were positively correlated with proinflammatory cytokine such as IL-23、IL-6、 IF-17、TNF-α、IL-1β and IFN-γ. The above results showed that the intestinal flora were closely related to the secretion and expression of cytokines in the body, and they interacted with each other to regulate immune function. Thus, Chrysanthemum polysaccharides could ameliorate ulcerative colitis by fostering beneficial intestinal flora growth, modulating the balance of intestinal microecology and restoring the immune system.

Biotransformation and metabolic profile of buddleoside with human intestinal microflora by ultrahigh-performance liquid chromatography coupled to hybrid linear ion trap/orbitrap mass spectrometer.

Buddleoside (also known as linarin) as the major flavonoid in Chrysanthemum morifolium Ramat., has been reported to possess a wide range of pharmacological activities. The human intestinal microbiota might have an important impact on drug metabolism and ultimately on the drug oral bioavailability. However, the interaction of the buddleoside with human intestinal bacteria remains unknown. In this study, the conversion of buddleoside by different bacteria from human feces was firstly investigated. A reliable, sensitive and rapid analytical method, ultra performance liquid chromatography was established and successfully applied to investigate the metabolites and metabolic profile of buddleoside by human intestinal bacteria. Among the isolated bacteria, four strains including Escherichia sp. 4, Escherichia sp. 34, Enterococcus sp. 45 and Bacillus sp. 46 showed more powerful conversion capability. Based on the accurate mass data and the characteristic MS(n) product ions, the parent and six metabolites were detected and tentatively identified compared with blank samples. The metabolites were produced by four main metabolic pathways including deglycosylation, acetylation, methylation and hydroxylation. Buddleoside could be firstly converted to its aglycon acacetin (M2) by the majority of the isolated intestinal bacteria. Subsequently, M2 was further metabolize to its methylated (M3), acetylated (M4), hydroxylated (M5) and hydrogenated product (M6). However, acacetin-7-glucosid (M1) was obtained only from the minor bacterial samples like Bacillus sp. 46. To further explain the metabolism of buddleoside, the β-d-glucosidase and α-l-rhamnosidase activities of four strains were analyzed. Bacillus sp. 46 could only produce α-l-rhamnosidase, while the other three strains showed two kinds of enzyme activities. Furthermore, the activities of α-l-rhamnosidase and β-d-glucosidase reached the highest level at 12-18h and 10-12h, respectively. The metabolic routes and metabolites of buddleoside produced by human intestinal microflora were firstly reported in this paper. The results will be very helpful for the further investigation of the pharmacokinetic research of buddleoside and to unravel how it works in vivo.

Investigation of the interactions between Chrysanthemum morifolium flowers extract and intestinal bacteria from human and rat.

Flos Chrysanthemi, dried flower of Chrysanthemum morifolium Ramat, has drawn much attention recently owing to its potential beneficial health effects for human. Flos Chrysanthemi products are usually taken orally and metabolized by intestinal microflora. However, there has been no investigation of the comprehensive metabolic profile of the Flos Chrysanthemi extract by intestinal flora owing to its chemical complexity and the limitations of analytical methods. In this paper, a rapid, sensitive and automated analysis method, ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry including MSE technology and automated data processing Metabolynx™ software, was developed and successfully applied for the biotransformation and metabolic profile of flavonoids in the Flos Chrysanthemi extract by intestinal flora from human and rat. A total of 32 metabolites were detected and tentatively identified in human and rat intestinal bacterial samples. These metabolites indicated that hydrolysis, hydroxylation, acetylation, methylation, hydrogenation and deoxygenation were the major conversion pathways of flavonoids in the Flos Chrysanthemi extract in vitro. Furthermore, the effects of the Flos Chrysanthemi extract on the growth of different intestinal bacteria were detected using an Emax precision microplate reader. Certain pathogenic bacteria such as Enterobacter, Enterococcus, Clostridium and Bacteroides were significantly inhibited by Flos Chrysanthemi, while commensal probiotics such as Lactobacillus and Bifidobacterium were moderately promoted. Our observation provided further evidence for the importance of intestinal bacteria in the metabolism and potential activity of the Flos Chrysanthemi extract. The results will also be helpful for the further pharmacokinetic study of Flos Chrysanthemi and to unravel how it works in vivo.

Peptidome characterization of the antipyretic fraction of Bubali Cornu aqueous extract by nano liquid chromatography with orbitrap mass spectrometry detection.

In the present study, the antipyretic activity of Bubali Cornu (water buffalo horn) fraction and its metabolomics were investigated. The fraction decreased rat rectal temperature, and 13 endogenous metabolites were identified as potential biomarkers. Selected metabolites were involved in arachidonic acid metabolism and glycerophospholipid metabolism etc. Following treatment with the fraction, four metabolites, pyroglutamic acid, palmitelaidic acid, leukotriene A4, and prostaglandin A2 were reversed. In addition, the levels of interleukin-1β, tumor necrosis factor-α, prostaglandin E2 , and cyclic adenosine monophosphate in plasma were also reversed after treatment as determined by enzyme linked immunosorbent assay. Furthermore, nano-flow liquid chromatography with orbitrap mass spectrometry detection was used to analyze the peptides in the fraction. In total, 824 peptide sequences mainly from keratins were determined, with Keratin 14, Keratin 34, and Keratin 86 representing the three main types of keratin hydrolysis in water buffalo horn based on peptide heat maps. Of the identified peptides, 81.2% were hydrophilic and the molecular weight of 70.3% of identified peptides was lower than 2000 Da. According to the metabolomics- and peptidomics-based approach used in the present study, it is feasible to identify and analyze the active peptide matrix from animal-horn-derived traditional Chinese medicines.