Isocorynoxeine (IC), a major alkaloid found in Uncaria rhynchophylla, exhibits wide beneficial effects on the cardiovascular and cardiocerebral vascular systems. Its metabolic pathway, however, has not been well studied yet. In this study, an ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (U-HPLC/Q-TOFMS) method was developed to investigate IC metabolism in plasma, urine and bile in rats given IC orally at 40 mg/kg.Nine male Wistar rats were given IC 40 mg/kg orally. Blood, urine and bile samples were collected at pre-specified times to measure the concentration of IC.A total of 35 metabolites were tentatively identified by the co-chromatography of biosamples and comparison of the retention time, characteristic molecular ions and fragment ions with those of the authentic standards or tentatively identified by MS/MS determination along with MassFragment software. Among them, 18, 33 and 18 metabolites were found in plasma, urine and bile samples, respectively. The relative percentage area of each metabolite was also determined to better understand the major metabolic pathways of IC in rats.The result indicates that IC undergoes extensive metabolism in vivo, mainly including hydrolysis, oxidation, isomerization, demethylation, epoxidation, reduction, glucuronidation, hydroxylation and N-oxidation, which is helpful for the further pharmacokinetic study of IC in vivo.

Tectorigenin (Te) is a main active component in the flowers of Pueraria thomsonii Benth. and the rhizomes of Belamcanda chinensis (L.) DC. Previously, we have reported the pharmacokinetic properties of Te in rat plasma. The purpose of this study was to investigate the urinary excretion of Te after oral administration to rats at different dose levels. Using UHPLC/Q-TOFMS, totally 26 metabolites were detected in rat urine after oral administration of Te at dose of 65 and 130 mg/kg. Among them, nine metabolites, Te, tectorigenin-7-O-glucuronide-4′-sulfate (Te-7G-4′S), tectorigenin-7-O-glucuronide (Te-7G), tectorigenin-7-O-sulfate (Te-7S), tectorigenin-4′-O-glucuronide (Te-4′S), isotectorigenin, genistein, irisolidone-7-O-glucuronide (Ir-7G), and irisolidone, were identified by comparing the retention time, UV and MS spectra with those of authentic standards. A UHPLC/Q-TOFMS method for simultaneous quantification and semi-quantification of all the metabolites in urine was developed. The cumulative urinary excretions of Te and the major metabolite Te-7G were 1.99 and 5.80 μmol at 65 mg/kg, 3.05 and 6.48 μmol at 130 mg/kg, accounted for 4.17 % and 15.8, 2.81 and 9.49 % of administrated Te, respectively. The excretion rates of Te-7G, Te-7G-4′S, Ir-7G, and Te reached a maximum between 12 and 24 h after oral dosing at 65 and 130 mg/kg. The cumulative urine excretion rates of Te were 23.1 and 20.1 % within 72 h at 65 and 130 mg/kg, respectively. These results suggested that the glucuronidation was the primary metabolic pathway especially at low dose level.

•This was the first report to evaluate PK of irisolidone.•UPLC/Q-TOF MS method evaluates pharmacokinetics of irisolidone and the main metabolites.•A total of 15 metabolites and 8 metabolic pathways of irisolidone were found in rats.Irisolidone, a major isoflavone found in Pueraria lobata flowers, exhibits a wide spectrum of bioactivities, while its metabolic pathways and the pharmacokinetics of its metabolites in vivo have not been investigated yet. In the present study, an ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method was employed to investigate the metabolic pathways of irisolidone and the pharmacokinetics of its main metabolites in rats, after a single 100 mg/kg oral dose of irisolidone. Protein precipitation method was used to prepare plasma samples. A total of 15 metabolites included irisolidone were detected and tentatively identified based on the mass spectral fragmentation patterns, elution order or confirmed using available reference standards. The pharmacokinetics of the main metabolites included three glucuronide metabolites tectorigenin-7-O-glucuronide (Te-7G), 6-hydroxybiochanin A-6-O-glucuronide (6-OH-BiA-6G), irisolidone-7-O-glucuronide (Ir-7G), and three sulfate metabolite tectorigenin-7-O-sulfate-4′-O-sulfate (Te-7S-4′S), tectorigenin-7-O-sulfate (Te-7S) and irisolidone-7-O-sulfate (Ir-7S), and aglycone tectorigenin (Te), and irisolidone (Ir) were evaluated. The plasma concentrations reached maximal values of 0.297 μmol/L at 10.3 h for Te-7S-4′S, 0.199 μmol/L at 21.7 h for Te-7G, 0.154 μmol/L at 8.00 h for Te-7S, 4.10 μmol/L at 15.3 h for 6-OH-BiA-6G, 10.7 μmol/L at 9.71 h for Ir-7G, 0.918 μmol/L at 11.3 h for Te, 0.150 μmol/L at 8.67 h for Ir-7S, and 0.843 μmol/L at 9.67 h for Ir, respectively. Since the total plasma concentrations of conjugated metabolites were much higher than that of the irisolidone aglycone, an extensive phase II metabolism plays an important role in the pharmacokinetics of irisolidone in vivo.

•A new method, microwave-assisted solution polymerization, has been developed for the synthesis of poly (d,l-lactide)-graft-pullulan (PL).•This technique can condense the time of synthesis from hours to minutes.•This method increased the yield of PL and lactide conversion.•This method can produce PL with different characteristics.•PL was applied for anti-cancer drug curcumin delivery as the forms of micelles and microparticles.A novel microwave-assisted method was developed to synthetize amphiphilic copolymer poly (d,l-lactide)-graft-pullulan (PL) in a monomode microwave reactor. The effects of microwave power, ratio of catalyst/lactide, ratio of lactide/hydroxyl group of pullulan (lactide/OH-P) and solvent on the synthesis were further investigated. Three samples (designated as PL 8, 9, and 6), characterized by FT-IR and NMR, were applied to form nanoparticles and microparticles investigated by dynamic light scattering, fluorescence spectroscopy and transmission electron microscopy. PL9 and PL6 were used for loading model drug curcumin. The results indicated that microwave-assisted synthesis shortened the copolymerization of PL, with higher yield and lactide conversion, from 24 h to 5 min and showed some specific microwave effects compared with conventional oil heating. PL with a relative higher substitution degree gave nanoparticles with smaller sizes and critical aggregation concentrations. The solubility of curcumin was increased to 1.97 mg mL−1 as the forms of nanoparticles.

•Screening for metabolites of kakkalide and irisolidone in intestinal bacteria.•UPLC/Q-TOF MS method was developed for metabolites identification.•Total 17 metabolites and 7 metabolic pathways were found in vitro.Kakkalide and irisolidone, the main isoflavones of Flos Puerariae, exhibit a wide spectrum of bioactivities. Intestinal bacteria biotransformation plays an important role in the metabolic pathways of flavones, and is directly related to the bioactivities of the prodrugs after oral administration. To the best of our knowledge, the metabolic pathways of kakkalide and irisolidone in vitro have not been comprehensively studied yet. This paper describes the strategy using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) for the rapid analysis of the metabolic profiles of kakkalide and irisolidone after incubated with human and rat intestinal bacteria. Bacteria incubated samples were prepared and analyzed after incubated under anaerobic conditions for 48 h. A total of 17 metabolites, including parent compounds, were detected in human and rat intestinal bacteria incubated samples. The results obtained indicate that hydrolysis, dehydroxylation, demethoxylation, demethylation, hydroxylation, decarbonylation, and reduction were the detected metabolic pathways of kakkalide and irisolidone in vitro. The conversion rate of irisolidone in human and rat bacteria was 8.57% and 6.51%, respectively. Biochanin A was the relatively main metabolite of irisolidone, and the content of biochanin A in human and rat bacteria was 3.68% and 4.25%, respectively. The conversion rate of kakkalide in human and rat bacteria was 99.92% and 98.58%, respectively. Irisolidone was the main metabolite of kakkalide, and the content of irisolidone in human and rat bacteria was 89.58% and 89.38%, respectively. This work not only provides the evidence of kakkalide and irisolidone metabolites in vivo, but also demonstrates a simple, fast, sensitive, and inexpensive method for identification of metabolites of other compounds transformed by intestinal bacteria.

•This is the first report to evaluate plasma pharmacokinetics of the conjugated metabolites in rats orally administered tectorigenin.•Eight conjugated metabolites of tectorigenin in rat plasma were simultaneously determined using U-HPLC/Q-TOFMS method.•The present study provides useful information for pharmacokinetic and pharmacodynamic investigations of isoflavones.Tectorigenin is a major isoflavone found in the flowers of Pueraria thomsonii Benth. and the rhizomes of Belamcanda chinensis (L.) DC. It possesses hepatoprotective, estrogenic, hypoglycemic and anti-inflammatory activities. In the present study, the plasma pharmacokinetic profile of tectorigenin in rats was evaluated. We developed a selective and accurate U-HPLC/Q-TOFMS method for the simultaneous characterization of nine tectorigenin metabolites, and quantitation of six major metabolites in rat plasma, including tectorigenin-7-O-glucuronide-4′-O-sulfate (Te-7G-4′S), tectorigenin-di-O-sulfate (Te-diS), tectorigenin-7-O-glucuronide (Te-7G), tectorigenin-4′-O-glucuronide (Te-4′G), tectorigenin-7-O-sulfate (Te-7S) and tectorigenin after oral administration of tectorigenin (130 mg/kg). The plasma concentrations reached maximal values of 6.20 ± 2.05 μmol/L at 0.96 ± 0.68 h for Te-7G-4′S, 4.42 ± 1.36 μmol/L at 1.92 ± 2.15 h for Te-diS, 33.50 ± 4.89 μmol/L at 0.75 ± 0.67 h for Te-7G, 3.28 ± 1.01 μmol/L at 0.75 ± 0.67 h for Te-4′G, 12.80 ± 2.80 μmol/L at 0.85 ± 1.54 h for Te-7S, and 12.0 ± 0.63 μmol/L at 0.23 ± 0.15 h for tectorigenin, respectively. Enterohepatic recirculation resulted in double or triple peaks concentration curve/time profiles of the metabolites. Since the total plasma concentrations of tectorigenin conjugated metabolites were much higher than that of the tectorigenin aglycone, an extensive phase II metabolism plays an important role in the pharmacokinetics of tectorigenin in vivo.

Irisolidone, a major isoflavone found in Pueraria lobata flowers, exhibits a wide spectrum of bioactivities, while its metabolic pathway in vivo has not been investigated. In this study, an ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method was employed to investigate the in vivo metabolism of irisolidone in rats. Plasma, bile, urine, and feces were collected from rats after a single 100 mg/kg oral dose of irisolidone. Protein precipitation, solid phase extraction (SPE) and ultrasonic extraction were used to prepare samples of plasma, bile/urine, and feces, respectively. A total of 46 metabolites were detected and tentatively identified based on the mass spectral fragmentation patterns, elution order or confirmed using available reference standards. The metabolic pathways of irisolidone in rats included decarbonylation, reduction, demethylation, demethoxylation, dehydroxylation, hydroxylation, sulfation, and glucuronidation. The relative content of each metabolite was also determined to help understand the major metabolic pathways of irisolidone in rats.

A multicomponent quantification fingerprint based on HPLC coupled with diode array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI/MS) technique has been established for the analysis of phenolic compounds in 12 samples originated from 5 different cultivars of Chrysanthemum morifolium flowers in China. Four caffeoylquinic acids and 15 flavonoids in the capitulum were identified by comparing the retention times and ultraviolet spectra as well as the mass spectrum and/or matching the empirical molecular formula with that of reference compounds, and the contents of these compounds have been determined simultaneously. The samples from three medicinal cultivars significantly differed in the quality and quantity of flavonoid aglycones and glycosides compared with those from two edible cultivars, which allows the possibility of showing the chemical distinctness of these cultivars and may be useful in their standardization. Moreover, the antiallergic effects of these cultivars were comparatively assayed for the first time. A representative medicinal cultivar, ‘huaiju’, showed potential activity on the inhibition of antigen-induced degranulation from RBL-2H3 cells and compound 48/80-induced scratching in mice, whereas the in vitro and in vivo antiallergic activities of two edible cultivars were weak. The results suggested that the quality and quantity of some active flavonoid aglycones should be responsible for the pharmacological profiles of these cultivars.

Tectoridin is a major isoflavone found in the flowers of Pueraria thomsonii Benth. It possesses estrogenic, hypoglycemic, anti-oxidant, and anti-inflammatory activities. In the present study, we evaluated the plasma pharmacokinetic profile of tectoridin in rats. We isolated a new metabolite, tectorigenin-7-O-glucuronide-4′-O-sulfate (Te-7G-4′S), from the bile of rats treated orally with tectoridin and determined its chemical structure by spectral analysis. Furthermore, we developed a selective and accurate method for the simultaneous quantification of tectoridin metabolites, including Te-7G-4′S, tectorigenin-7-O-glucuronide (Te-7G), tectorigenin-7-O-sulfate (Te-7S), and tectorigenin in rat plasma, and measured their plasma concentrations in rats orally administered tectoridin (200 mg/kg). Plasma concentrations of Te-7G-4′S, Te-7G, Te-7S, and tectorigenin reached maximal values of 21.4 ± 13.8 μmol at 3.50 ± 1.87 h, 20.5 ± 9.7 μmol at 3.17 ± 1.81 h, 14.3 ± 3.3 μmol at 5.58 ± 3.07 h, and 8.67 ± 3.07 μmol at 4.92 ± 2.87 h, respectively. Enterohepatic recirculation resulted in double peaks or a flat concentration curve/time profile of the metabolites. Since plasma concentrations of tectorigenin conjugated metabolites were higher than those of the tectorigenin aglycone, it can be concluded that extensive phase II metabolism plays an important role in the pharmacokinetics of tectoridin and tectorigenin in vivo.Highlights► A new glucuronide-sulfate diconjugate was isolated and structurally determined for the first time. ► Three conjugate metabolites were simultaneously identified and quantified in rat plasma by HPLC–DAD and LC/TOF/MS. ► It provides helpful information for the potential therapeutic application of tectoridin-containing phytopharmacueticals.

This study was undertaken to assess the plasma pharmacokinetic profile of kakkalide (KA), the major isoflavone found in extracts from the dried flower of Pueraria lobata. The main metabolites were identified using HPLC-DAD or LC/MS/MS method, and a HPLC-UV method for simultaneous quantification of the metabolites as well as the parent compound in plasma was developed. Rat plasma contained three glucuronide metabolites, irisolidone-7-O-glucuronide (Ir-7G), tectorigenin-7-O-glucuronide (Te-7G) and 6-OH biochanin A-glucuronide (6-OH BiA-G), as well as KA and trace amount of irisolidone (Ir) after oral administration of 200 mg/kg KA. The pharmacokinetics of KA and three glucuronide conjugates in rat plasma was determined for the first time using a simple, selective and accurate HPLC method. The AUC0−t values of the glucuronide metabolites are significantly greater than that of KA. They were detectable in rat plasma at different time points, indicating that glucuronidation during KA metabolism in vivo may occur in different sites, first in intestine and then in liver. Moreover, enterohepatic recirculation may result in the slow elimination of these glucuronide metabolites.

Two new isomeric alkaloids, 18,19-dehydrocorynoxinic acid B (1) and 18,19-dehydrocorynoxinic acid (2), were isolated from the CHCl3 extract of the leaves of Uncaria rhynchophylla, together with four known rhynchophylline-type alkaloids, corynoxeine (3), isocorynoxeine (4), rhynchophylline (5), and isorhynchophylline (6), and an indole alkaloid glucoside, vincoside lactam (7). The structures of compounds 1 and 2 were elucidated by spectroscopic methods including UV, IR, HREIMS, 1D and 2D NMR, and CD experiments. The activity assay showed that compounds 3−6, with a C-16 carboxylic ester group, and 7 exhibited inhibitory activity on lipopolysaccharide (LPS)-induced NO release in primary cultured rat cortical microglia (IC50: 13.7−19.0 μM). However, only weak inhibitory activity was observed for compounds 1 and 2, with a C-16 carboxylic acid group (IC50: >100 μM).

Isocorynoxeine (ICN) is one of the major bioactive tetracyclic oxindole alkaloids found in Uncaria rhynchophylla (Miq.) Jacks. that is widely used for the treatment of hypertension, vascular dementia, and stroke. The present study was undertaken to assess the plasma pharmacokinetic characteristics of major ICN metabolites, and the role of simulated gastric and intestinal fluid (SGF and SIF), human and rat liver microsomes (HLMs and RLMs), and seven recombinant human CYP enzymes in the major metabolic pathway of ICN. A rapid, sensitive and accurate UHPLC/Q-TOF MS method was validated for the simultaneous determination of ICN and its seven metabolites in rat plasma after oral administration of ICN at 40 mg/kg. It was found that 18.19-dehydrocorynoxinic acid (DCA) and 5-oxoisocorynoxeinic acid (5-O-ICA) were both key and predominant metabolites, rather than ICN itself, due to the rapid and extensive metabolism of ICN in vivo. The further study indicated that ICN was mainly metabolized in human or rat liver, and CYPs 2C19, 3A4 and 2D6 were the major enzymes responsible for the biotransformation of ICN to DCA and 5-O-ICA in human. These findings are of significance in understanding of the pharmacokinetic nature of tetracyclic oxindole alkaloids, and provide helpful information for the clinical co-administration of the herbal preparations containing U. rhynchophylla with antihypertensive drugs that are mainly metabolized by CYP3A4 and CYP2C19.Download high-res image (205KB)Download full-size image