The Metabolic Profiling of Isorhamnetin-3-O-Neohesperidoside Produced by Human Intestinal Flora Employing UPLC-Q-TOF/MS.

Isorhamnetin-3-O-neohesperidoside is the major active substance of Puhuang, a traditional herb medicine widely used in clinical practice to tackle many chronic diseases. However, little is known about the interactions between this ingredient and intestinal flora. In this study, ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry together with automated data analysis software (Metabolynx™) was used for analysis of the metabolic profile of isorhamnetin-3-O-neohesperidoside by the isolated human intestinal bacteria. The parent and three metabolites isorhamnetin-3-O-glucoside, isorhamnetin and quercetin were detected and identified based on the characteristics of their deprotonated molecules. These metabolites indicated that isorhamnetin-3-O-neohesperidoside was firstly deglycosylated to isorhamnetin-3-O-glucoside and subsequently to the aglycone isorhamnetin, and the latter was demethylated to quercetin. The majority of bacteria such as Escherichia sp. 23 were capable of converting isorhamnetin-3-O-neohesperidoside to considerable amounts of aglycone isorhamnetin and further to minor amounts of quercetin, while minor amounts of isorhamnetin-3-O-glucoside were detected in minority of bacterial samples such as Enterococcus sp. 30. The metabolic pathway and metabolites of isorhamnetin-3-O-neohesperidoside by the different human intestinal bacteria were firstly investigated. Furthermore, the metabolites of isorhamnetin-3-O-neohesperidoside might influence the effects of traditional herb medicines. Thus, our study is helpful to further unravel how isorhamnetin-3-O-neohesperidoside and Puhuang work in vivo.

Metabolic profiles of the Flos Abelmoschus manihot extract by intestinal bacteria from the normal and CKD model rats based on UPLC-Q-TOF/MS.

Flos Abelmoschus manihot is a traditional herbal medicine widely used in clinical practice to tackle chronic kidney disease (CKD) for thousands of years. Nowadays, many studies indicate that gut bacteria are closely related to the progression of CKD and CKD-related complications. In this study, a UPLC-Q-TOF/MS method coupled with the MetaboLynx™ software was established and successfully applied to investigate the metabolites and metabolic profile of Flos A. manihot extract by intestinal bacteria from normal and CKD rats. Eight parent components and eight metabolites were characterized by their protonated ions. Among these compounds, 15 were detected in the two group samples while M16 was only determined in the CKD model samples. Compared with the quercetin-type glycosides, fewer myricetin-type and gossypetin-type metabolites were obtained in the two group samples. These metabolites suggested that deglycosylation and methylation are the major metabolic pathways of Flos A. manihot extract. Few differences of metabolite classes were observed in the two group samples. However, the concentrations of aglycones such as quercetin, myricetin and gossypetin in the normal samples were notably higher than those in the CKD model samples. The results are important in unravelling the pharmacological effects of A. manihot and clarifying its mechanism of action in vivo.

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.

Comparative characterization of nucleotides, nucleosides and nucleobases in Abelmoschus manihot roots, stems, leaves and flowers during different growth periods by UPLC-TQ-MS/MS.

Nucleotides, nucleosides and nucleobases have been proven as important bioactive compounds related to many physiological processes. Abelmoschus manihot (L.) Medicus from the family of Malvaceae is an annual herbal plant of folk medicine widely distributed in Oceania and Asia. However, up to now, no detailed information could be available for the types and contents of nucleotides, nucleosides and nucleobases contained in A. manihot roots, stems, leaves as well as the flowers. In the present study, an UPLC-TQ-MS/MS method was established for detection of the twelve nucleotides, nucleosides and nucleobases. The validated method was successfully applied to identify the 12 analytes in different parts of A. manihot harvested at ten growth periods. 2'-deoxyinosine was not detected in all of the A. manihot samples. The data demonstrated that the distribution and concentration of the 12 compounds in A. manihot four parts were arranged in a decreasing order as leaf>flower>stem>root. Based on the results, the leaves and flowers of A. manihot could be developed as health products possessed nutraceutical and bioactive properties in the future. This method might also be utilized for the quality control of the A. manihot leaves and other herbal medicines being rich in nucleotides, nucleosides and nulecobases.

UPLC-Q-TOF/MS-based screening and identification of the main flavonoids and their metabolites in rat bile, urine and feces after oral administration of Scutellaria baicalensis extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese Medicines (TCMs) are increasingly used in combination with Western medicine. Scutellaria baicalensis Georgi (Lamiaceae) is a widely used TCM in treating various diseases. However, the in vivo metabolism of its main bioactive flavonoids, baicalin, baicalein, wogonoside and wogonin, needs further study. MATERIALS AND METHODS: A systematic method based on ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) technique combined with Metabolynx(TM) software was developed to speculate the metabolites and excretion profiles of the main flavonoids in S. baicalensis extract in rats bile, urine and feces samples after oral administration of the extract. RESULTS: Four parent components and a total of 15 metabolites were tentatively detected in vivo. All metabolites were detected including sulfate and glucuronide conjugates, hydroxylated, methylated, acetylated and deoxygenated products. Twelve metabolites were from the rat urine, five from the feces and two from the bile. Among them, several products were reported firstly. CONCLUSION: The research provided useful information for further study of the pharmacology and mechanism of action of S. baicalensis extract in vivo and a proposed method which could develop an integrated template approach to analyze screening and identification of biological samples after oral administration of TCMs.

Identification of the metabolites of myricitrin produced by human intestinal bacteria in vitro using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

OBJECTIVE: To investigate the metabolic routes and metabolites of myricitrin, an important active ingredient of traditional herbal medicine, yielded by the isolated human intestinal bacteria, which have not been reported previously. METHODS: Fresh human fecal samples were collected from a healthy female volunteer and about 100 different bacterial colonies were isolated. Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry technique combined with Metabolynx™ software was used for analysis of the metabolic profile of myricitrin by the isolated human intestinal bacteria. RESULTS: One hundred different bacterial colonies, which developed on plates, were picked up, and four of them were further identified by using the technique of 16S rRNA gene sequencing due to their relatively strong metabolic capacity toward myricitrin. Most of them belong to Escherichia. Parent compound and three metabolites (quercetin-3-O-rhamnoside, myricetin and quercetin) were detected in the isolated bacterial samples compared with blank samples. The metabolic pathways of myricitrin included deglycosylation and dehydroxylation. CONCLUSIONS: These metabolites suggested that myricitrin was first dehydroxylated to quercetin-3-O-rhamnoside and subsequently deglycosylated to quercetin. Additionally, myricitrin could also be deglycosylated to the aglycon myricetin. Moreover, those metabolites might influence the biological effect of myricitrin in vivo, which led to affect the clinical effects of the medicinal plants and traditional herb medicines.

Analysis of the metabolites of isorhamnetin 3-O-glucoside produced by human intestinal flora in vitro by applying ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Isorhamnetin 3-O-glucoside, which is widely contained in many vegetables and rice, is expected to be metabolized by intestinal microbiota after digestion, which brings about the profile of its pharmacological effect. However, little is known about the interactions between this active ingredient and the intestinal flora. In this study, the preculture bacteria and GAM (general anaerobic medium) broth with isorhamnetin 3-O-glucoside were mixed for 48 h of incubation. Ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry was used for analysis of the metabolites of isorhamnetin 3-O-glucoside in the corresponding supernatants of fermentation. The parent and five metabolites were found and preliminarily identified on the basis of the chromatograms and characteristics of their protonated ions. Four main metabolic pathways, including deglycosylation, demethoxylation, dehydroxylation, and acetylation, were summarized to explain how the metabolites were converted. Acetylated isorhamnetin 3-O-glucoside and kaempferol 3-O-glucoside were detected only in the sample of Escherichia sp. 12, and quercetin existed only in the sample of Escherichia sp. 4. However, the majority of bacteria could metabolize isorhamnetin 3-O-glucoside to its aglycon isorhamnetin, and then isorhamnetin was degraded to kaempferol. The metabolic pathway and the metabolites of isorhamnetin 3-O-glucoside yielded by different isolated human intestinal bacteria were investigated for the first time. The results probably provided useful information for further in vivo metabolism and active mechanism research on isorhamnetin 3-O-glucoside.

Ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry for determination of avicularin metabolites produced by a human intestinal bacterium.

Intestinal bacteria from human were screened to isolate the specific bacteria involved in the metabolism of avicularin. A Gram-positive anaerobic bacterium, strain 46, capable of metabolizing avicularin (quercetin-3-O-arabinoside) was isolated for the first time. Its 16S rRNA gene sequence showed 99% similarity with that of Bacillus. Then strain 46 was identified as a species of the genus Bacillus, and was named to be Bacillus sp. 46. Additionally, the metabolites were analyzed by ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) technique combined with Metabolynx™ software. The structure of these metabolites were proposed and confirmed by comparing the UPLC retention time and MS/MS spectrum with that of authentic standards. Parent compound and six metabolites were detected in the isolated bacterial samples compared with blank samples. Avicularin (M1) was anaerobic metabolized to its aglycone quercetin (M2) and methoxylated avicularin (M3, M4), then quercetin was converted to quercetin glycosides: quercetin-3-O-rhamnoside (M5), quercetin-3-O-glucoside (M6) and quercetin-7-O-glucoside (M7) by Bacillus sp. 46. The metabolic pathway and metabolites of avicularin by the intestinal bacterium Bacillus sp. 46 were reported for the first time.