Bioactivities and chemical profiling comparison and metabolomic variations of polyphenolics and steroidal glycoalkaloids in different parts of Solanum nigrum L.

INTRODUCTION: Solanum nigrum L. is a traditional medicinal herb and edible plant. Many studies provide evidence that S. nigrum L. is a nutritious vegetable. Polyphenols and steroidal glycoalkaloids are the main components. OBJECTIVES: This study aimed to systemically evaluate the phytochemical profile, quantification, and bioactivities of polyphenolics and glycoalkaloids in different parts of S. nigrum L. RESULTS: Total polyphenols (TPC) and total glycoalkaloids (TGK) were determined using the Folin-Ciocalteu and acid dye colorimetric methods, respectively. A total of 55 polyphenolic constituents (including 22 phenolic acids and 33 flavonoids) and 24 steroidal glycoalkaloids were identified from different parts using ultrahigh-performance liquid chromatography Q-exactive high-resolution mass spectrometry (UHPLC-QE-HRMS), of which 40 polyphenols (including 15 phenolic acids and 25 flavonoids) and one steroidal glycoalkaloid were characterised for the first time in S. nigrum L. Moreover, typical polyphenols and glycoalkaloids were determined using HPLC-UV and HPLC-evaporative light-scattering detector (ELSD), respectively. In addition, the TPC and TGK and their typical constituents were compared in different anatomical parts. Finally, the antioxidant capacities of polyphenolic extracts from different parts of S. nigrum L. were evaluated by ·OH, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and ferric-reducing antioxidant power (FRAP) assay in vitro. In addition, the antitumour effects of TGK from different parts of S. nigrum L. on the proliferation of PC-3 cells were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Polyphenolic and glycoalkaloid extracts from different parts of S. nigrum L. showed different antioxidant and cytotoxic capacities in vitro. CONCLUSION: This is the first study to systematically differentiate between polyphenolic and glycoalkaloid profiles from different parts of S. nigrum L.

Pharmacokinetics, oral bioavailability and metabolic analysis of solasodine in mice by dried blood spot LC-MS/MS and UHPLC-Q-Exactive MS.

Solasodine, a major ingredient in Solanaceae family, has various biological functions such as inducing neurogenesis, anticonvulsant and anti-tumor. Its risk assessment has also drawn public attention. However, little is known about its oral bioavailability and metabolic process. In this study, an liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of solasodine in mice dried blood spot (DBS) samples. To block nonspecific adsorption, DBS samples were pretreated with bovine serum albumin (BSA) and then extracted with ethyl acetate. This method was applied to a pharmacokinetic and bioavailability study of solasodine. The absolute bioavailability was only 1.28%. Thereafter, its metabolites in mice were characterized using an ultra-performance liquid chromatography Q-Exactive high-resolution mass spectrometer (UHPLC-QE-HRMS). Several isomeric metabolites were well separated and differentiated using their retention time, fragmentation pathways and correspondingly fragmentation rules of solasodine. As a result, 21 metabolites were characterized including 16 phase I and 5 phase II metabolites. The proposed metabolic pathways showed that solasodine mainly experienced oxidation, dehydration, dehydrogenation and sulfation. These results could help us to better understand the efficacy and safety of solasodine.

Comprehensive metabolic profiling of Alismatis Rhizoma triterpenes in rats based on characteristic ions and a triterpene database.

Alismatis Rhizoma (AR) is widely used in Chinese medicine, and its major bioactive components, triterpenes, reportedly possess various pharmacological activities. Therefore, it is very important to study the metabolism of triterpenes in vivo. However, the metabolism of AR triterpene extract has not been comprehensively elucidated due to its complex chemical components and metabolic pathways. In this study, an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method, which was based on the characteristic ions from an established database of known triterpenes, was used to analyze the major metabolites in rats following the oral administration of Alismatis Rhizoma extracts (ARE). As a result, a total of 233 constituents, with 85 prototype compounds and 148 metabolites, were identified for the first time. Hydrogenation, oxidation, sulfate and glucuronidation conjugation were the major metabolic pathways for triterpenes in AR. In addition, the mutual in vivo transformation of known ARE triterpenes was discovered and confirmed for the first time. Those results provide comprehensive insights into the metabolism of AR in vivo, which will be useful for future studies on its pharmacodynamics and pharmacokinetics. Moreover, this established strategy may be useful in metabolic studies of similar compounds.

Bovine Lactoferrin Quantification in Dairy Products by a Simple Immunoaffinity Magnetic Purification Method Coupled with High-Performance Liquid Chromatography with Fluorescence Detection.

This study described a simple, specific, and sensitive method using immunoaffinity magnetic purification coupled with high-performance liquid chromatography-fluorescence (HPLC-FL) detection for determination of bovine lactoferrin (bLf) in dairy products. BLf was selectively extracted from dairy products using immunoaffinity beads and then detected by HPLC-FL with its intrinsic fluorescence. During the analysis, standard solutions of bLf were pretreated with Tween 20, an anti-adsorptive agent, for blocking the nonspecific binding of bLf to polypropylene tubes. The calibration curve was linear over the range of 0.8-30 µg mL-1. The validated method was successfully applied to measure bLf at the intact level in dairy products.

Metabolic Stability and Metabolite Characterization of Capilliposide B and Capilliposide C by LC⁻QTRAP⁻MS/MS.

Capilliposide B (LC-B) and Capilliposide C (LC-C), two new triterpenoid saponins extracted from Lysimachia capillipes Hemsl, exhibit potential anticancer activity both in vitro and in vivo. However, their metabolic process remains unclear. In this study, the metabolic stability of LC-B, LC-C, and Capilliposide A (LC-A, a bioactive metabolite of LC-B and LC-C) was investigated in human, rat, and mouse liver microsomes, respectively. Thereafter, their metabolites were identified and characterized after oral administration in mice. As a result, species difference was found in the metabolic stability of LC-B and LC-C. All three compounds of interest were stable in human and rat liver microsomes, but LC-B and LC-C significantly degraded in mouse liver microsomes. The metabolic instability of LC-B and LC-C was mainly caused by esterolysis. Moreover, 19 metabolites were identified and characterized in mouse biological matrices. LC-B and LC-C mainly underwent deglycosylation and esterolysis, accompanied by dehydration, dehydrogenation, and hydroxylation as minor metabolic reactions. Finally, the metabolic pathway of LC-B and LC-C in mice was proposed. Our results updated the preclinical metabolism and disposition process of LC-B and LC-C, which provided additional information for better understanding efficacy and safety.

A targeted strategy to identify untargeted metabolites from in vitro to in vivo: Rapid and sensitive metabolites profiling of licorice in rats using ultra-high performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry.

It is challenging to conduct in vivo metabolic study for traditional Chinese medicines (TCMs) because of complex components, unpredictable metabolic pathways and low metabolite concentrations. Herein, we proposed a sensitive strategy to characterize TCM metabolites in vivo at an orally clinical dose using ultra-high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry (UHPLC-QTRAP-MS). Firstly, the metabolism of individual compounds in rat liver microsomes was studied to obtain the metabolic pathways and fragmentation patterns. The untargeted metabolites in vitro were detected by multiple ion monitoring-enhanced product ion (EPI) and neutral loss-EPI scans. Subsequently, a sensitive multiple reaction monitoring-EPI method was developed according to the in vitro results and predicted metabolites to profile the in vivo metabolites. Licorice as a model herb was used to evaluate and validate our strategy. A clinical dose of licorice water extract was orally administered to rats, then a total of 45 metabolites in urine, 21 metabolites in feces and 35 metabolites in plasma were detected. Among them, 18 minor metabolites have not been reported previously and 6 minor metabolites were first detected in vivo. Several isomeric metabolites were well separated and differentiated in our strategy. These results suggested that this new strategy could be widely used for the detection and characterization of in vivo metabolites of TCMs.

Tissue distribution of capilliposide B, capilliposide C and their bioactive metabolite in mice using liquid -tandem mass spectrometry.

Lysimachia capillipes Hemsl (Primulaceae), a folk medicinal plant in China, showed significant anti-tumor activities in vivo and in vitro. Capilliposide B (LC-B) and capilliposide C (LC-C) are the main bioactive components in this plant. To explore their tissue distribution, a reliable bioanalytical method for the quantification of LC-B, LC-C and their bioactive metabolite, capilliposide A (LC-A), in mouse tissues was developed and validated. In this study, the tissue distribution profiles of the three compounds were examined after intravenous administration of pure LC-B and oral administration of total saponins of L. capillipes Hemsl extract (LCE) for 10 days. Method validation was conducted over the curve range 10.0-5000 ng/mL for all three analytes in various tissue homogenates. The relative standard deviation of intra-day and inter-day precision of the QC samples was <14.7%, and the accuracy ranged from 85.9 to 114.0%. The results indicated that LC-B was rapidly and widely distributed throughout the whole body except for muscle following intravenous administration of LC-B. In addition, LC-A was only in liver, intestine, lung and stomach. After oral administration of LCE, LC-B and LC-C were distributed into various tissues. The highest levels were observed in stomach and intestine.

Optimization of solid-phase extraction and liquid chromatography-tandem mass spectrometry for simultaneous determination of capilliposide B and its active metabolite in rat urine and feces: Overcoming nonspecific binding.

Capilliposide B, a novel oleanane triterpenoid saponin isolated from Lysimachia capillipes Hemsl, showed significant anti-tumor activities in recent studies. To characterize the excretion of Capilliposide B, a reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of Capilliposide B and its active metabolite, Capilliposide A in rat urine and feces. Sample preparation using a solid-phase extraction procedure was optimized by acidification of samples at various degrees, providing extensive sample clean-up with a high extraction recovery. In addition, rat urinary samples were pretreated with CHAPS, an anti-adsorptive agent, for overcoming nonspecific analytes adsorption during sample storage and process. The method validation was conducted over the curve range of 10.0-5000ng/ml for both analytes. The intra- and inter-day precision and accuracy of the QC samples showed ≤11.0% RSD and -10.4-12.8% relative error. The method was successfully applied to an excretion study of Capilliposide B following intravenous administration.

An integrated approach for profiling oxidative metabolites and glutathione adducts using liquid chromatography coupled with ultraviolet detection and triple quadrupole-linear ion trap mass spectrometry.

The use of liquid chromatography (LC) coupled with triple quadrupole linear ion trap (Qtrap) mass spectrometry (MS) for both quantitative and qualitative analysis in drug metabolism and pharmacokinetic studies is of great interest. Here, a new Qtrap-based analytical methodology for simultaneous detection, structural characterization and semi-quantitation of in vitro oxidative metabolites and glutathione trapped reactive metabolites was reported. In the current study, combined multiple ion monitoring and multiple reaction monitoring were served as surveying scans to trigger product ion spectral acquisition of oxidative metabolites and glutathione adduct, respectively. Then, detection of metabolites and recovery of their MS/MS spectra were accomplished using multiple data mining approaches. Additionally, on-line ultraviolet (UV) detection was employed to determine relative concentrations of major metabolites. Analyses of metabolites of clozapine and nomifensine in rat liver microsomes not only revealed multiple oxidative metabolites and glutathione adducts, but also identified their major oxidative metabolism and bioactivation pathways. The results demonstrated that the LC/UV/MS method enabled Qtrap to perform the comprehensive profiling of oxidative metabolites and glutathione adducts in vitro.

Development and validation of an LC-MS/MS method for simultaneous quantification of levodopa and MD01 in rat plasma and its application to a pharmacokinetic study of mucuna pruriens extract.

Mucuna pruriens, an ancient Indian herbal medicine containing levodopa, is widely used for Parkinson's disease. In order to simultaneously determine levodopa and 1,1-dimethyl-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (MD01) in rat plasma, an improved LC-MS/MS method was developed and validated for a pharmacokinetic study in rats orally administered levodopa or Mucuna pruriens extract (MPE). Elimination of matrix effect and improvement of extraction recovery were achieved through systematic optimization of reversed-phase and hydrophilic interaction chromatographic conditions together with sample clean-up procedures. A satisfactory chromatographic performance was obtained with a Thermo Aquasil C18 column (50 × 2.1 mm, 3 µm) using acetonitrile and water containing 0.2% formic acid as mobile phases. Futhermore, sodium metabisulfite and formic acid were used as stabilizers in neat solutions as well as rat plasma. The method was validated in a dynamic range of 20.0-10,000 ng/mL for levodopa and MD01; the intra- and inter-day precision and accuracy were acceptable. The method was successfully utilized to determine the levodopa level in plasma samples of rats administered levodopa or MPE. Pharmacokinetic results showed that an increase in the AUC of levodopa was observed in rats following oral administration of multiple doses of MPE. Copyright © 2016 John Wiley & Sons, Ltd.

Cell-based assays in combination with ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry for screening bioactive capilliposide C metabolites generated by rat intestinal microflora.

Many plant-derived glycosides are used as medications. It is common that these glycosides show poor intestinal absorption but their metabolites generated by intestinal microflora demonstrate strong bioactivity. Hence, it is crucial to develop a method for the identification and characterization of the metabolites, and consequently reveal the pathway in which the glycosides are processed in gut. In this study, cell-based assays in combination with ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) were developed for rapid discovery and evaluation of the metabolites of a glycoside compound, capilliposide C (LC-C). 92.7% of LC-C was biotransformed by rat intestinal microflora after 36-h incubation at 37°C. Human cancer cell lines HepG2, PC-3 and A549 was treated with metabolites pool, respectively, which was followed by cell viability assays and characterization of metabolites using UHPLC-QTOF-MS/MS. As a result, significant cytotoxicity was observed for the metabolites pool, from which six metabolites were identified. Based on the metabolites identified, deglycosylation and esterolysis were proposed as the major metabolic pathways of LC-C in rat intestinal microflora. In addition, M4, an esterolysis product of LC-C, was obtained and evaluated for its bioactivity in vitro. As a result, M4 exhibited a reduction in cell viability in HepG2 with an IC50 value of 17.46±1.55µg/mL.

Determination of thailandepsin B in rat plasma with a LC-MS/MS method.

BACKGROUND: Thailandepsin B (TDP-B) is a potent histone deacetylase inhibitor under development. A reliable bioanalytical method for the quantification of TDP-B in plasma samples is required. RESULTS: The stabilizer mixture containing hydrochloric acid, formic acid and dichlorvos was applied to stabilize TDP-B in matrix samples. The method validation was conducted over the curve range of 1.00 to 1000 ng/ml. The intrabatch and interbatch precision and accuracy of the quality control samples showed ≤10.9% relative standard deviation and -6.7% to 15.0% relative error. Moreover, a possible metabolite M of TDP-B was tentatively characterized. CONCLUSION: A reliable LC-MS/MS method was developed and validated for the quantification of TDP-B and was successfully applied to a rat pharmacokinetic study.

Simultaneous determination of capilliposide B and capilliposide C in rat plasma by LC-MS/MS and its application to a PK study.

BACKGROUND: Lysimachia capillipes Hemsl (Primulaceae), a folk medicinal plant in China, showed significant anti-tumor activity in recent studies. A reliable LC-MS/MS method was developed and validated for the simultaneous determination of capilliposide B and capilliposide C, the major bioactive components in this plant, in rat plasma. RESULTS: Rat plasma and whole blood samples were pretreated with dichlorvos, an esterase inhibitor, minimizing degradation of analytes in biological samples. The method validation was conducted over the curve range of 10.0 to 5000 ng/ml for both analytes. The intra- and inter-day precision and accuracy of the QC samples showed ≤6.1% RSD and 1.3-3.7% relative error. CONCLUSION: The method was successfully applied to determine the concentrations of capilliposide B and capilliposide C in incurred rat plasma samples, after administration of Lysimachia capillipes Hemsl extract for a rat PK study.

HPLC method for the simultaneous determination of four compounds in rat plasma after intravenous administration of Portulaca oleracea L. extract

The objective of the present study was to develop a simple and selective HPLC method for the simultaneous determination of hesperidin (HP), caffeic acid (CA), ferulic acid (FA) and p-coumaric acid (p-CA) in rat plasma after intravenous administration of Portulaca oleracea L. extract (POE). With the hyperoside as the internal standard, the sample pretreatment procedure involved simple single-step extraction with methanol of 0.2 mL plasma. The mobile phase consisted of methanol-acetonitrile-tetrahydrofuran-0.5% glacial acetic acid (5:3:18:74, v/v/v/v). The calibration curves were linear over the range of 0.1-25 µg mL-1, 0.1-25 µg mL-1, 0.1-25 µg mL-1and 0.015-3 µg mL-1 for HP, CA, FA and p-CA, respectively. The method developed was suitable for the pharmacokinetic study of HP, CA, FA and p-CA in rats after intravenous administration of POE.

O objetivo do estudo foi desenvolver um método simples e específico de HPLC para a determinação simultânea de hesperidina (HP), ácido caféico (CA), ácido ferúlico (FA) e ácido p-cumárico (p-CA) em plasma de rato após a administração intravenosa de extrato Portulaca oleracea L. (POE) empregando hyperosídeo como padrão interno de referência. Metanol foi empregado para os analitos em plasma (0,2 mL). A fase móvel isocrática foi composta por metanol-acetonitrila-tetraidrofurano-0,5% ácido acético glacial (5:3:18:74, v/v/v/v). Curvas de calibração foram lineares na faixa de concentração de 0,1-25 µg mL-1, 0,1-25 µg mL-1, 0,1-25 µg mL-1 e 0,015-3 µg mL-1 para HP, CA, FA e p-CA, respectivamente. O método desenvolvido foi adequado para estudo farmacocinético de HP, CA, FA e p-CA em ratos após a administração intravenosa de POE.

LC determination and pharmacokinetic study of the main phenolic components of Portulaca oleracea L. extract in rat plasma after oral administration.

This study investigated the pharmacokinetics of hesperidin (HP), ferulic acid (FA) and p-coumaric acid (CA) in rat plasma after oral administration of Portulaca oleracea L. extract (POE). The plasma concentrations were determined by HPLC with vitexin-2″-O-rhamnoside (VR) as internal standard. The calibration curves were linear over the range 0.1-5 µg mL(-1), 0.1-5 µg mL(-1)and 0.015-3 µg mL(-1) for HP, FA and CA, respectively. The validated method was suitable to the pharmacokinetic study of HP, FA and CA in rats after oral administration at a single dose of POE.

HPLC determination of five polyphenols in rat plasma after intravenous administration of hawthorn leaves extract and its application to pharmacokinetic study.

A simple and specific HPLC-UV method was developed to simultaneously determine five active compounds including vitexin-4"-O-glucoside (VG), vitexin-2"-O-rhamnoside (VR), vitexin (VIT), rutin (RUT) and hyperoside (HP) in rat plasma after intravenous administrating the hawthorn leaves extract (HLE). With baicalin as internal standard (I.S.), sample pretreatment involved a one-step extraction with methanol of 0.2 ml plasma. The HPLC assay was carried out using a Phenomsil C18 analytical column with UV detection at 332 nm. The mobile phase consisted of methanol-acetonitrile-tetrahydrofuran-1% glacial acetic acid (6:1.5:18.5:74, v/v/v/v). The calibration curves were liner over the range of 2.030-500.5, 0.1513-75.64, 0.2507-12.54, 0.5128-25.64 and 0.4032-20.16 µg/ml for VG, VR, VIT, RUT and HP, respectively. The relative standard deviations (RSD) of the intra- and inter-day precisions for the analysis of the five analytes were between 1.0 and 8.9% with accuracies (relative error) below 8.2% for the analysis of the five analytes. The average extraction recoveries of five analytes were more than 82.67 ± 4.74%. The HPLC method herein described was fully validated and successfully applied to the pharmacokinetic studies after intravenous administration of HLE solution to rats over three doses.