Identification of the Metabolites in Rat Urine after Oral Administration and Elucidation of the Metabolic Process of Naozhenning Granule Using LC-MS.

Naozhenning (NZN) granule, a Chinese herbal formula, is widely used to treat craniocerebral trauma and promote functional recovery. In our previous study, the chemical components, as well as the serum metabolites in the male Sprague-Dawley rats of the NZN granule after oral administration were characterized. In this study, the urine metabolites in the male Sprague-Dawley rats were further investigated by ultrahigh-performance liquid chromatography-Q Exactive hybrid quadrupole-Orbitrap high-resolution accurate mass spectrometry. In order to identify the urine metabolites comprehensively, three sample preparation methods were used, including solid-phase extraction, protein precipitation method and solvent partition. Based on the accurate molecular weight and the fragmentation information from the MS spectra, a total of 76 urine metabolites were identified, which including 17 prototypes and 59 metabolites. The results showed that the detected urine metabolites were different for the different pretreatment methods, as some metabolites could only be detected in the particular pretreatment method. In addition, the metabolic processes of the components from NZN granule to the serum and urine were also elucidated and discussed. The results will provide useful information for further studying the relationship between the chemical components and pharmacological activity of NZN granule.

A novel insight into the potential toxicity mechanisms of Zhi-Zi-Hou-Po decoction by dynamic urinary metabolomics based on UHPLC-Q-Exactive Orbitrap-MS.

In recent years, depression occurs frequently. Given the long duration of the disease and the high risk of recurrence, the treatment of depression requires long-term medication. Zhi-Zi-Hou-Po Decoction (ZZHPD) has been used in clinical treatment of depression and related diseases for many years, and the potential toxic damage caused by its long-term use has gradually emerged. Existing research methods that expose toxicity by a one-time administration of large doses cannot provide a reference for clinical safe drug use. In this study, the potential toxicity of ZZHPD in repeated administration was studied by urinary metabolomics with nondestructive sampling. Based on ultra-high performance liquid chromatography-quadruple-Exactive Orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS) and chemometrics, 33 differential biomarkers, such as 3-hydroxybutyric acid, indole sulfuric acid, hippuric acid and citric acid, were screened and dynamically tracked. The changes of some endogenous substances showed obvious time dependence. Further analysis of these time-dependent components in combination with network pharmacology revealed that the potential hepatotoxicity and nephrotoxicity of ZZHPD were related to the disorders of amino acid metabolism, energy metabolism, lipid metabolism, nucleotide metabolism and gut microflora metabolism pathway. This study can better grasp the occurrence and development of drug toxicity, and provide reference for rational and safe drug use and potential toxicity prevention of ZZHPD.

UPLC-Q-TOF/MS-based metabolic profiling comparison of four major bioactive components in normal and CKD rat plasma, urine and feces following oral administration of Cornus officinalis Sieb and Rehmannia glutinosa Libosch herb couple extract.

Cornus officinalis-Rehmannia glutinosa herb couple is widely used herb medicine in clinical practice to treat chronic kidney disease (CKD). However, the in vivo integrated metabolism of its main bioactive components in CKD rats remains unknown. In this study, UPLC-Q-TOF/MS technique combined with Metabolynx™ software, was developed and successfully applied for analysis of metabolic profiles of the bioactive components of the herb couple in normal and CKD rat biological samples. Main parent components of the herb couple extract such as loganin, morroniside and catalpol were absorbed into the blood circulation of the normal and CKD rats. Another parent component acteoside was almost completely degraded. Seventeen metabolites involved in the in vivo metabolism processes were tentatively identified. These metabolites indicated that loganin was mainly metabolized to the demethylated product, and morroniside was firstly deglycosylated to the aglycone and the latter was subsequently demethylated and acetylated. Additionally, hydrogenation and deglycosylation were the principal metabolic reactions of catalpol; while O-glucuronide and O-sulphate conjugates were observed as major metabolites for methylated caffeic acid and hydroxytyrosol released from acteoside. Compared with the normal group, the CKD rat showed lower conversion capability. Few kinds and minor amounts of the metabolites appeared in the CKD rat samples. While considerable amounts of the parent compounds were detected in the CKD plasma. This will help maintain a high blood drug concentration which might be beneficial for the treatment of CKD. The proposed method could develop an integrated template approach to analyze screening and identification of the bioactive components in plasma, urine and feces after oral administration of herb medicines. Additionally, this investigation might provide helpful chemical information for further pharmacology and active mechanism research on herb medicines.

Main Human Urinary Metabolites after Genipap (Genipa americana L.) Juice Intake.

Genipap (Genipa americana L.) is a native fruit from Amazonia that contains bioactive compounds with a wide range of bioactivities. However, the response to genipap juice ingestion in the human exposome has never been studied. To identify biomarkers of genipap exposure, the untargeted metabolomics approach in human urine was applied. Urine samples from 16 healthy male volunteers, before and after drinking genipap juice, were analyzed by liquid chromatography⁻high-resolution mass spectrometry. XCMS package was used for data processing in the R environment and t-tests were applied on log-transformed and Pareto-scaled data to select the significant metabolites. The principal component analysis (PCA) score plots showed a clear distinction between experimental groups. Thirty-three metabolites were putatively annotated and the most discriminant were mainly related to the metabolic pathways of iridoids and phenolic derivatives. For the first time, the bioavailability of genipap iridoids after human consumption is reported. Dihydroxyhydrocinnamic acid, (1R,6R)-6-hydroxy-2-succinylcyclohexa-2,4-diene-1-carboxylate, hydroxyhydrocinnamic acid, genipic acid, 12-demethylated-8-hydroxygenipinic acid, 3(7)-dehydrogenipinic acid, genipic acid glucuronide, nonate, and 3,4-dihydroxyphenylacetate may be considered biomarkers of genipap consumption. Human exposure to genipap reveals the production of derivative forms of bioactive compounds such as genipic and genipinic acid. These findings suggest that genipap consumption triggers effects on metabolic signatures.

High Resolution Mass Spectrometric Analysis of Secoiridoids and Metabolites as Biomarkers of Acute Olive Oil Intake-An Approach to Study Interindividual Variability in Humans.

SCOPE: Phenolic compounds are minor components of extra virgin olive oil (EVOO). Secoiridoids are the major components contributing to the phenolic content of EVOO. Information is lacking regarding their potential as biomarkers for EVOO intake. METHODS AND RESULTS: Healthy volunteers (n = 9) ingested 50 mL of EVOO in a single dose containing 322 mg kg-1 total phenolic content (caffeic acid equivalents) and 6 mg 20 g-1 hydroxytyrosol and its derivatives. Plasma is collected before (0 h) and at 0.5, 1, 2, 4, and 6 h after ingestion. Urine samples are collected prior to ingestion (0 h) and at 0-4, 4-8, 8-15, and 15-24 h. Samples are analyzed by UPLC coupled with an Exactive Orbitrap MS. Partial least squares discriminant analysis with orthogonal signal correction is applied to screen for metabolites that allow sample discrimination. Plasma biomarkers and urine biomarkers are selected although individual variability is observed among volunteers. Results are in accordance with in vitro experiments performed (in vitro digestion and hepatic microsomal activity assays). CONCLUSIONS: Plasma (elenolic acid + H2 ; p-HPEA-EA + H2 + glucuronide) and urinary (3,4-DHPEA-EA, 3,4-DHPEA-EA + H2 +glucuronide, methyl 3,4-DHPEA-EA + H2 +glucuronide) secoiridoid compounds are selected as biomarkers to monitor EVOO intake showing good predictive ability according to multivariate analysis.

Targeted and Untargeted Metabolomics to Explore the Bioavailability of the Secoiridoids from a Seed/Fruit Extract (Fraxinus angustifolia Vahl) in Human Healthy Volunteers: A Preliminary Study.

The bark, seeds, fruits and leaves of the genus Fraxinus (Oleaceae) which contain a wide range of phytochemicals, mostly secoiridoid glucosides, have been widely used in folk medicine against a number of ailments, yet little is known about the metabolism and uptake of the major Fraxinus components. The aim of this work was to advance in the knowledge on the bioavailability of the secoiridoids present in a Fraxinus angustifolia Vahl seed/fruit extract using both targeted and untargeted metabolomic analyses. Plasma and urine samples from nine healthy volunteers were taken at specific time intervals following the intake of the extract and analyzed by UPLC-ESI-QTOF. Predicted metabolites such as tyrosol and ligstroside-aglycone glucuronides and sulfates were detected at low intensity. These compounds reached peak plasma levels 2 h after the intake and exhibited high variability among the participants. The ligstroside-aglycone conjugates may be considered as potential biomarkers of the Fraxinus secoiridoids intake. Using the untargeted approach we additionally detected phenolic conjugates identified as ferulic acid and caffeic acid sulfates, as well as hydroxybenzyl and hydroxyphenylacetaldehyde sulfate derivatives which support further metabolism of the secoiridoids by phase I and (or) microbial enzymes. Overall, the results of this study suggest low uptake of intact secoiridoids from a Fraxinus angustifolia Vahl extract in healthy human volunteers and metabolic conversion by esterases, glycosidases, and phase II sulfo- and glucuronosyl transferases to form smaller conjugated derivatives.

Bioavailability of phenolics from an oleuropein-rich olive (Olea europaea) leaf extract and its acute effect on plasma antioxidant status: comparison between pre- and postmenopausal women.

PURPOSE: Preclinical studies suggest a potential protective effect of oleuropein in osteoporosis, and one of the proposed mechanisms is the modulation of the oxidative stress. Oleuropein bioavailability and its effect on antioxidant status in pre- and postmenopausal women are unknown. The aim of the present study was to investigate the oral bioavailability of an olive leaf extract rich in oleuropein (40 %) and its effect on antioxidant status in postmenopausal women compared to premenopausal women. METHODS: Premenopausal (n = 8) and postmenopausal women (n = 8) received 250 mg of olive leaf extract, blood samples (t = 0, 1, 2, 3, 4, 6, 8, 12, 16 and 24 h) were taken, and 24-h urine divided into five fractions was collected. Olive-leaf-extract-derived metabolites were analyzed in plasma and urine by HPLC-ESI-QTOF and UPLC-ESI-QqQ, and pharmacokinetics parameters were determined. Ferric reducing antioxidant ability and malondialdehyde levels were measured in plasma. RESULTS: Plasma levels of hydroxytyrosol glucuronide, hydroxytyrosol sulfate, oleuropein aglycon glucuronide and oleuropein aglycon derivative 1 were higher in postmenopausal women. MDA levels were significantly decreased (32%) in postmenopausal women and inversely correlated with hydroxytyrosol sulfate levels. Postmenopausal women excreted less sulfated metabolites in urine than premenopausal women. CONCLUSIONS: Our results suggest that postmenopausal women could be a target population for the intake of olive phenolics in order to prevent age-related and oxidative stress-related processes such as osteoporosis.

Identification of metabolites of geniposide in rat urine using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Geniposide, an iridoid glycoside, is an important and characteristic compound in the fruits of Gardenia jasminoides Ellis, a commonly used medicinal herb in Chinese traditional and folk medicine for the treatment of inflammation and jaundice. However, few studies have been carried out on the metabolism of geniposide. In this study, we have established a rapid and sensitive method using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/ESI-QTOF-MS) for analysis of the metabolic profile of geniposide in rat urine after oral administration. A total of ten metabolites were detected and identified by comparing their fragmentation patterns with that of geniposide using Metabolynx™ and MassFragment™ software tools. The results revealed that the principal metabolism pathways of geniposide in rat occurred after deglycosylation of the irdoid glycoside take place and this is followed by glucuronidation and the pyran-ring cleavages. The major metabolite, the glucuronic acid conjugate of genipin as observed in vivo, was further confirmed by the in vitro enzymatic study. The results of this work have demonstrated the feasibility of the UPLC/ESI-QTOF-MS approach for rapid and reliable characterization of metabolites from iridoid compounds.

Determination of sweroside in rat plasma and bile for oral bioavailability and hepatobiliary excretion.

Sweroside is an active ingredient of iridoid glycoside isolated from the flower buds of Lonicera japonica THUNB. A quantitative HPLC-UV method was developed for monitoring sweroside in rat plasma, urine, feces and bile. The method was successfully applied for a basic pharmacokinetic study. The obtained data of pharmacokinetics were applied to evaluate the oral bioavailabilities of sweroside and the active ingredients of purified herbal extracts (IGEs-1). The absolute bioavailability was estimated to F(sweroside) 0.31% and F(IGEs-1) 0.67%. The majority of sweroside excreted to feces revealed one reason of the low oral bioavailability. The values of F(IGEs-1) much higher than that of F(sweroside) reveals that ingredients in IGEs-1 such as loganin, secoxyloganin and some phenolic acids may promote the absorption of sweroside. The study of hepatobiliary excretion was achieved by an in vivo microdialysis sampling method after intravenous administration of sweroside. The percentage of accumulation of free form sweroside in bile duct was 31.2+/-7.2% of the total dosage. It may be one reason why sweroside possesses strong hepatoprotective effect.

Analysis of iridoids from Harpagophytum and eleutherosides from Eleutherococcus senticosus in horse urine.

LC/ESI-MS n methods have been previously set up to detect the administration of (i) Harpagophytum and (ii) preparations containing a plant capable of anti-stress properties: Eleutherococcus senticosus. Harpagoside has been found to be the main indicator of Harpagophytum administration in the horse. These methods have been applied to a large number of horse urine samples of various origins. Regarding the detection of Harpagophytum administration, harpagoside, harpagide and 8-para-coumaroyl harpagide were detected together in only one sample out of 317. Eleutheroside E was found to be the main indicator of Eleutherococcus senticosus administration. It was detected in post-administration samples collected from two horses having received a feed supplement containing Eleutherococcus senticosus for several days. Out of the 382 samples tested, eleutheroside E was found in an unexpected large number of urine samples (39%) of various origins and its presence cannot be only due to the sole use of herbal dietary supplements.

Determination of geniposide in rat urine after oral administration of the traditional Chinese medicinal preparation yin-zhi-ku decoction by high-performance liquid chromatography.

A high-performance liquid chromatography method with solid-phase extraction is introduced for the determination of geniposide in rat urine after oral administration of yin-zhi-ku decoction. Geniposide and an internal standard (paeoniflorin) are extracted from urine using Strata cartridges. Analysis of the extract is then performed on a reversed-phase C18 column using acetonitrile-water (14:86, v/v) as eluting solvent system. UV detection is set at 238 nm. The calibration curve for geniposide is linear (r = 0.9996) in the concentration range of 2.0-240 microg/mL. Both intra- and interday precision of the geniposide are determined, and their relative standard deviation does not exceed 10%. The validated method is successfully applied to determine geniposide from rat urine after oral administration of yin-zhi-ku decoction.