In vitro and in vivo metabolic activation of rhein and characterization of glutathione conjugates derived from rhein.

Rhein (RH), 4,5-dihydroxyanthrauinone-2-carboxylic acid, is found in rhubarb (Dahuang), a traditional herbal medicine. RH has reportedly demonstrated multiple pharmacologic properties. Previous studies have also shown that RH induced hepatotoxicity, but the mechanisms of the adverse effect remain unknown. The major objective of the present study was to study the metabolic pathways of RH in order to identify potential reactive metabolites. One mono-hydroxylation metabolite (M1) was detected in urine and bile of rats given RH. M1 was also observed in rat and human liver microsomal incubations after exposure to RH. A total of three (GSH) conjugates (M2, M3 and M5) were detected in bile of rats treated with RH. We concluded that M2-M3 were directly derived from parent compound RH through spontaneous reaction with GSH. M5 was derived from M1 by reaction with GSH, which required cytoslic GSTs. M5 was further metabolized to the corresponding NAC conjugate (mercapturic acid) and was excreted in urine. P450 2C9 was mainly involved in the oxidation of RH.

UPLC-QTOF-MS Identification of the Chemical Constituents in Rat Plasma and Urine after Oral Administration of Rubia cordifolia L. Extract.

An effective ultra-performance liquid chromatography coupled with the quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF/MS) method was developed for analysing the chemical constituents in rat plasma and urine after the oral administration of Rubia cordifolia L. extract. Under the optimized conditions, nine of 11 prototypes in rat plasma and four prototypes in urine were identified or characterized by comparing the retention time, accurate mass, fragmentation patterns, reference compounds, and literature data. In total, six metabolites, including alizarin-1-O-ß-glucuronide, alizarin-2-O-ß-glucuronide, alizarin-1-O-sulfation, alizarin-2-O-sulfation, purpurin-1-O-ß-glucuronide, and purpurin-3-O-ß-glucuronide, were identified in rat plasma, which were confirmed by lavaging standard solutions. Purpurin was found to be able to be transformed into alizarin based on the results in which alizarin was detected in rat plasma after the oral administration of a purpurin solution. In total, four metabolites were found in rat urine, but their chemical structures were not confirmed. The results indicate that the metabolic pathway of alizarin involves glucuronidation and sulfation, with the purpurins having undergone glucuronidation. The components absorbed into the blood, and the metabolites have the opportunity to become bioactive constituents. The experimental results would supply a helpful chemical basis for further research on the mechanism of actions of Rubia cordifolia L.

Application of a highly sensitive magnetic solid phase extraction for phytochemical compounds in medicinal plant and biological fluids by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

A highly sensitive method using reduced graphene oxide with iron oxide (rGO/Fe3 O4 ) as the sorbent in magnetic SPE has been developed for the purification of five anthraquinones (emodin, rhein, aloeemodin, physcion, and chrysophanol) in rhubarb and rat urine by ultra-HPLC coupled with quadrupole TOF/MS. The extraction was accomplished by adding trace amount rGO/Fe3 O4 suspension to 200 mL of aqueous mixture, and the excellent adsorption capacity of the nanoparticles was fully demonstrated in this procedure. Under the optimized conditions, the calibration curves were linear in the concentration range of 0.05-27.77 ng/mL with correlation coefficients varying from 0.9902 to 0.9978. The LODs ranged from 0.28 to 58.99 pg/mL. The experimental results indicated that the proposed method was feasible for the analysis of anthraquinones in rhubarb and urine samples.

Identification of chemical constituents and rat metabolites of Kangxianling granule by HPLC-Q-TOF-MS/MS.

A high-performance liquid chromatography coupled with quadrupole time-of-flight mass tandem mass spectrometry method was established to characterize the chemical constituents of Kangxianling granule (KXL), a traditional Chinese medicine formula, and the metabolic profile in rat urine and plasma after oral administration of KXL. A total of 27 compounds in KXL extract and 13 prototype compounds with 12 metabolites in rat urine and plasma were identified. Among the 27 detected compounds, 15 were identified by comparing the retention time and MS data with that of reference compounds and the other 12 compounds were tentatively assigned based on the MS data and reference literature. The main prototype components absorbed in rat were amygdalin, salvianolic acid B, tanshinones and anthraquinones. Hydroxylation, glucuronidation and sulfation were the principal metabolic pathways in rat. The results revealed that the 25 compounds identified in rat urine and plasma were the potential active ingredients of KXL, which provides helpful chemical information for further study of the pharmacology mechanism of KXL.

In vivo metabolism study of rhubarb decoction in rat using high-performance liquid chromatography with UV photodiode-array and mass-spectrometric detection: a strategy for systematic analysis of metabolites from traditional Chinese medicines in biological samples.

High-performance liquid chromatography with diode-array detection (HPLC-DAD) and tandem mass spectrometry (HPLC-MS/MS) was used for separation and identification of metabolites in rat urine, bile and plasma after oral administration of rhubarb decoction. Based on the proposed strategy, 91 of the 113 potential metabolites were tentatively identified or characterized. Besides anthraquinones metabolites, gallic acid, (-)-epicatechin and (+)-catechin metabolites were also detected and characterized in these biological samples. Our results indicated that glucuronidation and sulfation were the main metabolic pathways of anthraquinones, while methylation, glucuronidation and sulfation were the main metabolic pathways of gallic acid, (-)-epicatechin and (+)-catechin. Phase I reactions (e.g., hydroxylation and reduction) played a relatively minor role compared to phase II reactions in metabolism of phenolic compounds of rhubarb decoction. The identification and structure elucidation of these metabolites provided essential data for further pharmacological and clinical studies of rhubarb and related preparations. Moreover, the results of the present investigations clearly indicated the relevance and usefulness of the combination of chromatographic, spectrophotometric, and mass-spectrometric analysis to detect and identify metabolites.

Mixed hemimicelles SPE based on CTAB-coated Fe3O4/SiO2 NPs for the determination of herbal bioactive constituents from biological samples.

In this paper, a solid-phase extraction (SPE) method based on mixed hemimicelles of cetyltrimethyl ammonium bromide (CTAB) on silica-coated magnetic nanoparticles (MNPs) is developed for extraction and preconcentration of compounds from the biological samples. We selected rhein and emodin which are the major active anthraquinones of rhubarb as model analytes. A high performance liquid chromatography-fluorescence detection (HPLC/FLD) method was developed for the determination of rhein and emodin in urine and serum samples. The main factors influencing the extraction efficiency including the amount of surfactant, the concentration of MNPs, the shaking time and the desorption ability of organic solvents were investigated and optimized. No interferences were caused by proteins or endogenous compounds in urine and serum samples. Good linearities (r(2)>0.9995) for all calibration curves were obtained, and the limits of detection (LODs) for rhein and emodin were 0.2 and 0.5 ng/mL in urine samples and 7 and 10 ng/mL in serum samples, respectively. Satisfactory recoveries (92.76-109.90% and 97.53-107.72% for rhein and emodin) in the biological matrices were achieved.

Simultaneous determination by UPLC-ESI-MS of scoparone, capillarisin, rhein, and emodin in rat urine after oral administration of Yin Chen Hao Tang preparation.

A simple, sensitive, and validated method was developed for simultaneous determination of scoparone, capillarisin, rhein, and emodin in rat urine by ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry (UPLC-MS). The urinary samples were analyzed on an Acquity UPLC BEH C18 1.7 microm 2.1x50 mm column. Scoparone, capillarisin, rhein, and emodin in rat urine were simultaneously analyzed with good separation. The lower limits of detection were 6.0, 9.0, 7.0, and 3.0 ng/mL, and the lower limits of quantification were 20.0, 33.0, 24.0, and 12.0 ng/mL for scoparone, capillarisin, rhein, and emodin, respectively. The intra- and inter-day precisions (RSD) were less than 9%. The intra- and inter-accuracies were found to be in the range of 94.14-104.54% for scoparone, 101.72-107.34% for capillarisin, 95.24-103.59% for rhein, and 101.32-107.82% for emodin at three concentration levels. The absolute recoveries for scoparone, capillarisin, rhein, and emodin were not less than 77.0%. The developed method has been applied to determine scoparone, capillarisin, rhein, and emodin in rat urine after oral administration of Yin Chen Hao Tang preparation, a traditional Chinese medicine formulation widely used in China for treatment of jaundice and liver disorders.

Screening procedure for detection of stimulant laxatives and/or their metabolites in human urine using gas chromatography-mass spectrometry after enzymatic cleavage of conjugates and extractive methylation.

A gas chromatography-mass spectrometry (GC-MS)-based screening procedure was developed for the detection of stimulant laxatives and/or their metabolites in human urine after enzymatic cleavage of conjugates followed by extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by capillary GC and identified by computerized MS in the full scan mode. By use of mass chromatography with the ions m/z 305, 290, 335, 320, 365, 350, 311, 326, 271, and 346, the possible presence of stimulant laxatives and/or their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra. This method allowed the detection of the diphenol laxatives bisacodyl, picosulfate, and phenolphthalein and of the anthraquinone laxatives contained in plant extracts and/or their metabolites in human urine samples. The overall recoveries of the stimulant laxatives and/or their metabolites ranged between 33% and 89% with a coefficient of variation of less than 15%, and the limits of detection ranged between 10 and 25 ng/mL (S/N 3) in the full scan mode. After ingestion of the lowest therapeutic dose of sodium picosulfate, its main metabolite, bisacodyl diphenol, was detectable in urine samples for 72 hours. After ingestion of the lowest therapeutic dose of a senna extract, the main metabolite of sennosides, rhein, was detectable in urine samples for 24 hours. This procedure is part of a systematic toxicological analysis procedure for acidic drugs and poisons with the modification of enzymatic cleavage of conjugates.

Pharmacokinetics of rhein from Onpi-to, an Oriental herbal medicine, in rats.

Onpi-to, an herbal medicine composed of five crude drugs (Rhei Rhizoma, Glycyrrhizae Radix, Ginseng Radix, Zingiberis Rhizoma and Aconiti Tuber), was administered orally to rats. Onpi-to includes 1.240% of total potential rhein derived from sennoside A, sennoside B, rhein 8-O-glucopyranoside and rhein. Plasma, urinary and biliary levels of rhein were determined by an HPLC-UV method. The plasma levels displayed curves characterized by maximum peaks at 8.3+/-5.2 min, 8.3+/-5.2 min and 20.0+/-21.9 min following dosages of 125, 250 and 500 mg/kg with mean concentrations of 1302.5+/-926.4, 2973.6+/-684.3 and 3118.8+/-1701.2 ng/ml, respectively, followed by a subsequent decline. Area under the concentration-time curve (AUC)(0-48 h) at doses of 125, 250 and 500 mg/kg were 752.3+/-321.5, 2443.3+/-554.4 and 4443.2+/-2641.3 ng.h/ml, respectively. In female rats, rhein plasma levels showed curves which had a maximum peak at 45.0+/-16.4 min after a dosage of 250 mg/kg with mean concentration of 3058.0+/-1533.7 ng/ml, followed by a subsequent decline. AUC(0-48 h) was 5537.7+/-1876.0 ng.h/ml. The cumulative urinary excretion of rhein and of conjugated rhein was 3.14+/-1.56% and 38.21+/-18.87% of dose, respectively, 48 h after dosing at 500 mg/kg of Onpi-to in male rats. The cumulative biliary excretion of rhein was 1.34+/-0.44% of dose 48 h after dosing at 500 mg/kg of Onpi-to in male rats.

[Analysis of anthraquinones in serum and urine after oral administration of semen Cassiae].

OBJECTIVE: To analysie the anthraquinones absorbed into serum by different animals (or human beings) after oral administration of Semen Cassiae. METHOD: Anthraquinones in serum and urine of rats and urine of healthy men after taking Semen Cassiae orally were detected with HPLC. RESULT: Only some of the anthraquinones were absorbed into serum. There were differences in absorption and metabolism of anthraquinones between rats and men and some new anthraquinones were produced in the process. CONCLUSION: Anthraquinones absorbed into serum by the experimental animals or men should become target for researching into active compounds of anthraquinones in Semen Cassiae.

Chromatographic identification of phenolic compounds in human urine following oral administration of the herbal medicines Daisaiko-to and Shosaiko-to.

Chemical identification of the compounds in human urine following administration of the traditional Chinese medicines, Daisaiko-to and Shosaiko-to (Dachaihu-tang and Xiaochaihu-tang in Chinese, respectively), was achieved by using a linear relationship between the logarithm of the capacity factor, log k', and that of the volume fraction of CH3CN, log X(s)(vol), in the aqueous mobile phase: -log k'=A+B log X(s)(vol). Comparison of the slope, B, and the intercept, A, between the urinary compound and its suspected authentic specimen gave satisfactory results in the chemical identification. We applied this method to the initial stage of pharmacokinetic studies on the herbal medicines and identified seven flavonoids and two anthraquinone derivatives in the urine specimens obtained after herbal administration.

Effects of Cassia obtusifolia (sicklepod) extracts and anthraquinones on muscle mitochondrial function.

Naturally occurring quinones and quinone-containing extracts of seeds of the toxic plant Cassia obtusifolia (sicklepod) affected muscle mitochondrial function. Aqueous suspensions and organic extracts of C. obtusifolia seeds slightly elevated plasma creatine kinase levels of Sprague-Dawley rats. These extracts were analyzed by fused silica capillary gas chromatography and found to contain nine anthraquinones and three anthrones. Urinary metabolites primarily consisted of beta-glucuronide conjugates of the anthraquinones. The three anthrones or conjugate analogues were not present in the urine in detectable amounts. Emodin, doxorubicin and organic extracts of C. obtusifolia inhibited NADH:cytochrome c oxidoreductase activity of bovine heart mitochondrial particles and NADH:CoQ oxidoreductase activity of porcine heart mitochondrial NADH dehydrogenase, whereas juglone was stimulatory. Relative quinone metabolism correlated with semiquinone formation rate and with redox potential. A protective effect of coenzyme Q against enzyme inhibition by anthraquinones was also observed.

Comparative physiological disposition of some anthraquinone glycosides and aglycones.

The in vitro microbial degradation and the urinary excretion and biliary secretion in rats of two anthraquinone glycosides (sennosides A and B) and four aglycones (sennidins A and B, rhein, and danthron) were studied using a high performance liquid chromatographic system with gradient elution and amperometric detection. Microbial degradation of sennosides A and B occurred almost exclusively in the presence of mice caecum inoculae and was associated with the release of sennidins A and B. Rhein and danthron were indiscriminately metabolized by bacteria sampled from all regions of mice intestine, whereas sennidins lacked stability in biological media. The fraction of the dose administered orally to rats and recovered as aglycones or as glucuronides in bile and urine after 48 hours was five times greater for rhein (15 per cent) and danthron (13.4 per cent) than for sennosides A (1.8 per cent) and B (2.8 per cent) excreted or secreted as sennidins. These results support the concept that anthraquinone glycosides are less likely to enter the systemic circulation and, thus, are able to exert their laxative effect at lower doses than aglycones.

A screening method for establishing laxative abuse.

Abuse of laxatives, most of them belonging to the group of colonic stimulants or cathartics, can cause various disorders. Extensive diagnostic work can be avoided by early toxicological screening of the suspected patients with respect to laxatives. Because no screening method of this kind was available, we developed a procedure with which all phenolic and anthraquinone laxatives--except sodium picosulfate--can be detected in urine. This method is based on high-performance thin-layer chromatography in two systems after pretreatment of a 20-mL urine sample with beta-glucuronidase and subsequent column extraction. The procedure is very sensitive: at least 32 h after a single dose of bisacodyl, danthron, phenolphthalein, or sennoside, the drug can be detected in the urine. Bisoxatin and oxyphenisatin are still detectable in the urine 18 h after intake. The method is also highly specific; none of 73 other drugs interfered in either of the two chromatographic systems. This procedure can be helpful for the early diagnosis of laxative abuse.