Analysis of five active ingredients of Er-Zhi-Wan, a traditional Chinese medicine water-honeyed pill, using the biopharmaceutics classification system.

Er-Zhi-Wan (EZW) is a traditional Chinese medicine with many clinical applications and used as a health product in East Asia. Five active ingredients (salidroside, specnuezhenide, nuezhenoside, luteolin, and oleanolic acid) were screened out from EZW to develop an in vitro rapid evaluation method for the classification of in vivo drug absorption behavior by biopharmaceutics classification system (BCS). Ultra-performance liquid chromatography was used for quantitative analysis. Solubility and permeability were assayed by equilibrium solubility and multiple models: everted rat intestinal sac model, cultured Caco-2 cells, octanol-water partition coefficient (LogP) method. The BCS properties of drugs were predicted using software applications, and the correlations of measured and predicted values of factors affecting oral drug absorption were calculated. The results were verified by measuring the absolute bioavailability of the active ingredients. Salidroside, specnuezhenide, and nuezhenoside were classified as BCS class III drugs, and luteolin was classified as a BCS class III/I drug because of the difference in LogP and intestinal permeability. Oleanolic acid was classified as a BCS class II/IV drug in acidic media and BCS class I/III drug in other media. Overall, EZW may be classified as a BCS class III drug, and permeability was identified as the primary factor limiting absorption. The results provide a novel method for the evaluation of the in vivo absorption of oral traditional Chinese medicines.

Pharmacokinetics of harpagoside in horses after intragastric administration of a Devil's claw (Harpagophytum procumbens) extract.

Devil's claw is used for the treatment of inflammatory symptoms and degenerative disorders in horses since many years, but without the substantive pharmacokinetic data. The pharmacokinetic parameters of harpagoside, the main active constituent of Harpagophytum procumbens DC ex Meisn., were evaluated in equine plasma after administration of Harpagophytum extract FB 8858 in an open, single-dose, two-treatment, two-period, randomized cross-over design. Six horses received a single dose of Harpagophytum extract, corresponding to 5 mg/kg BM harpagoside, and after 7 days washout period, 10 mg/kg BM harpagoside via nasogastric tube. Plasma samples at certain time points (before and 0-24 hr after administration) were collected, cleaned up by solid-phase extraction, and harpagoside concentrations were determined by LC-MS/MS using apigenin-7-glucoside as internal standard. Plasma concentration-time data and relevant parameters were described by noncompartmental model through PKSolver software. Harpagoside could be detected up to 9 hr after administration. Cmax was found at 25.59 and 55.46 ng/ml, t1/2 at 2.53 and 2.32 hr, respectively, and tmax at 1 hr in both trials. AUC0-inf was 70.46 and 117.85 ng hr ml-1 , respectively. A proportional relationship between dose, Cmax and AUC was observed. Distribution (Vz /F) was 259.04 and 283.83 L/kg and clearance (CL/F) 70.96 and 84.86 L hr-1  kg-1 , respectively. Treatment of horses with Harpagophytum extract did not cause any clinically detectable side effects.

UPLC-Q-TOF-MS and UPLC-MS/MS methods for metabolism profiles and pharmacokinetics of major compounds in Xuanmai Ganjie Granules.

Xuanmai Ganjie Granules (XMGJ), a widely used Chinese herbal formula in the clinic, is used for treatment of sore throats and coughs. Despite the chemical constituents having been clarifying by our previous studies, both of the metabolism and pharmacokinetic studies of XMGJ are unclear. This study aimed to explore the disposition process of XMGJ in vivo. A sensitive and selective ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) method was developed to analyze the absorbed components and metabolites in rat plasma and urine after oral administration of XMGJ. A total of 42 absorbed components, including 16 prototype compounds and 26 metabolites, were identified or tentatively characterized in rat plasma and urine after oral administration of XMGJ. Moreover, the pharmacokinetic studies of five compounds of XMGJ were investigated using ultra-high liquid chromatography with tandem mass spectrometry method. The results indicated that liquiritin, harpagoside, glycyrrhetic acid, liquiritigenin, formononetin and their metabolites might be the major components involved in the pharmacokinetic and metabolism process of XMGJ. This research showed a comprehensive investigation of XMGJ in vivo, which could provide a meaningful basis for further material basis and pharmacological as well as toxicological research.

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.

[Studies on effects of Achyranthes bidentata on tongsaimai pellets main active ingredients chlorogenic acid, isoliquiritin, harpagoside and glycyrrhizin in vivo pharmacokinetics].

To study on the effects of Achyranthes bidentata on Tongsaimai pellets main active ingredients chlorogenic acid, isoliquiritin, harpagoside and glycyrrhizin in rats in vivo pharmacokinetic behaviors, a method for the simultaneous determination of chlorogenic acid, isoliquiritin, harpagoside and liquiritigenin in rat plasma was established by UPLC-MS/MS. The analysis was performed on a waters Acquity BEH C18 column (2.1 mm x 100 mm, 1.7 microm) with the mixture of acetonitrile and 0.1% formic acid/water as mobile phase, and the gradient elution at a flow rate of 0.3 mL x min(-1). The analytes were detected by tandem mass spectrometry with the electrospray ionization (ESI) source and in the multiple reaction monitoring (MRM) mode. It turned out that the analytes of Tongsaimai pellets groups C(max) and AUC(Q-infinity) values were higher than that with A. bidentata group, and the C(max) values of chlorogenic acid had significantly difference (P < 0.05), the AUC(0-infinity) values of chlorogenic acid and glycyrrhizin had significantly difference (P < 0.05); The T(max) and CL values of two groups had no significantly difference. Results showed that the established method was specific, rapid, accurate and sensitive for the studies of Tongsaimai pellets four main active ingredients in rat in vivo pharmacokinetic, and A. bidentata have varying degrees of effects on Tongsaimai pellets four main active ingredients in rat in vivo pharmacokinetic behaviors.

[Absorption and pharmacokinetics of radix rehmanniae in rats].

In this paper, absorption and pharmacokinetic study of Radix Rehmanniae was studied by liquid chromatography coupled with mass spectrometry method after oral administration to rats. By comparing the chromatograms of ultraviolet, full scan, extracted ion and selective reaction monitoring (SRM) of standard solution, Radix Rehmanniae, blank plasma and rat plasma post drug administration, catalpol and ajugol were found to be the main compounds absorbed from Radix Rehmanniae. Plasma concentrations of aucubin, dihydrocatalpol, rehmannioside A (or rehmannioside B/ melittoside) and rehmannioside D were very low. Quantitative method for catalpol and aucubin and semi-quantitative method for other compounds in rat plasma were established. The pharmacokinetic study of those absorbed components was conducted after oral administration of 6 g x kg(-1) Radix Rehmanniae water extract to rats. Cmax, t(1/2) and AUC(0-infinity) of catalpol and ajugol were (2349.05 +/- 1438.34) and (104.25 +/- 82.05) ng x mL(-1), (0.86 +/- 0.32) and (0.96 +/- 0.37) h, (4407.58 +/- 2734.89) and (226.66 +/- 188.38) ng x h x mL(-1), respectively. tmax was at 1.00 h for catalpol and ajugol. Both catalpol and ajugol were absorbed and excreted rapidly.

Validated LC-MS method for simultaneous quantitation of catalpol and harpagide in rat plasma: application to a comparative pharmacokinetic study in normal and diabetic rats after oral administration of Zeng-Ye-Decoction.

A simple and efficient liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for simultaneous quantitation of catalpol and harpagide in normal and diabetic rat plasma. Protein precipitation extraction with acetonitrile was carried out using salidroside as the internal standard (IS). The LC separation was performed on an Elite C18 column (150 × 4.6 mm, 5 µm) with the mobile phase consisting of acetonitrile and water within a runtime of 12.0 min. The analytes were detected without endogenous interference in the selected ion monitoring mode with positive electrospray ionization. Calibration curves offered satisfactory linearity (r > 0.99) at linear range of 0.05-50.0 µg/mL for catalpol and 0.025-5.0 µg/mL for harpagide with the lower limits of quantitation of 0.05 and 0.025 µg/mL, respectively. Intra- and inter-day precisions (RSD) were <9.4%, and accuracy (RE) was in the -6.6 to 4.9% range. The extraction efficiencies of catalpol, harpagide and IS were all >76.5% and the matrix effects of the analytes ranged from 86.5 to 106.0%. The method was successfully applied to the pharmacokinetic study of catalpol and harpagide after oral administration of Zeng-Ye-Decoction to normal and diabetic rats, respectively.

Pharmacokinetics of 8-O-acetylharpagide and harpagide after oral administration of Ajuga decumbens Thunb extract in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ajuga decumbens Thunb is a medicinal plant native to China popularly used to treat chronic pelvic inflammation and hysteromyoma. Its main bioactive components are iridoid glycosides, such as 8-O-acetylharpagide and harpagide that had presented antibacterial, anti-inflammatory, and antiviral activities. AIM OF THE STUDY: To establish a sensitive LC-MS/MS method and compare the pharmacokinetics of 8-O-acetylharpagide and harpagide in rats after oral administration of their pure forms and from compounds obtained from Ajuga decumbens extract. MATERIALS AND METHODS: Rats received orally 15 mg/kg (equivalent of 6 mg/kg 8-O-acetylharpagide and 1.5mg/kg harpagide), 30 mg/kg and 60 mg/kg of Ajuga decumbens Thunb extract and were compared to animals that received 12 mg/kg of 8-O-acetylharpagide or 3mg/kg of harpagide p.o. Concentrations of 8-O-acetylharpagide and harpagide in plasma were determined by LC-MS/MS method at different time points and all pharmacokinetic parameters were estimated by non-compartmental analysis. RESULTS: Results showed that the iridoid glycosides were quickly absorbed by oral route and showed a dose-dependence profile. Pharmacokinetic parameters of both glycosides were essentially the same except Tmax when dosed as the extract or pure forms. CONCLUSION: 8-O-acetylharpagide was metabolized to harpagide, which affected the pharmacokinetic profiles of harpagide when dosed as the extract. This pharmacokinetic study seems to be useful for a further clinical study of Ajuga decumbens Thunb extract.

An in vivo microdialysis measurement of harpagoside in rat blood and bile for predicting hepatobiliary excretion and its interaction with cyclosporin A and verapamil.

Harpagoside, a major bioactive iridoid glucoside in genus Scrophularia, has been widely used in clinical practice for the treatment of pain in the joints and lower back for its neuroprotective and anti-inflammation activities. To investigate the pharmacokinetics and hepatobiliary excretion, an in vivo microdialysis method coupled with high performance liquid chromatography was developed to monitor the concentration of harpagoside in blood and bile. The harpagoside bile-to-blood distribution ratio (AUC(bile)/AUC(blood)) up to 986.28+/-78.46 significantly decreased to 6.41+/-0.56 or 221.20+/-18.92 after co-administration of cyclosporin A or verapamil. The results indicated that harpagoside went through concentrative elimination from the bile which was probably regulated by P-glucoprotein, providing possible clinical trials of co-administration of transporter inhibitors to decrease drug efflux, thus to enhance the curative effects.

Simultaneous determination of harpagoside and cinnamic acid in rat plasma by liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic studies.

A simple and sensitive method was developed for the simultaneous quantification of harpagoside and cinnamic acid in rat plasma using high-performance liquid chromatography system coupled to a negative ion electrospray mass spectrometric analysis. The plasma sample preparation was a simple deproteinization by the addition of two volumes of acetonitrile. The analytes were separated on an Intersil C8-3 column (2.1 mm i.d.x250 mm, 5 microm) with acetonitrile-5 mm ammonium formate aqueous solution (60:40, v/v) as mobile phase at a flow-rate of 0.2 mL/min. Detection was performed on a quadrupole mass spectrometer equipped with electrospray ionization (ESI) source operated under selected ion monitoring (SIM) mode. [M+HCOO]- at m/z 539 for harpagoside, [M-H]- at m/z 147 for cinnamic acid and [M-H]- at m/z 137 for salylic acid (internal standard) were selected as detecting ions, respectively. The method was validated over the concentration range 7-250 ng/mL for harpagoside and 5-500 ng/mL for cinnamic acid. The lower limits of quantitation for harpagoside and cinnamic acid were 7 and 5 ng/mL, respectively. The intra- and inter-day precisions (RSD%) were within 9.5% and the assay accuracies (RE%) ranged from -5.3 to 3.0% for both analytes. Their average recoveries were greater than 86%. Both analytes were proved to be stable during all sample storage, preparation and analysis procedures. The method was successfully applied to the pharmacokinetic study of harpagoside and cinnamic acid following oral administration of Radix Scrophulariae extract to rats.

Simultaneous determination of harpagoside and cinnamic acid in rat plasma by high-performance liquid chromatography: application to a pharmacokinetic study.

Radix Scrophulariae (xuanshen) is one of the famous Chinese herbal medicines widely used to treat rheumatism, tussis, pharyngalgia, arthritis, constipation, and conjunctival congestion. Harpagoside and cinnamic acid are the main bioactive components of xuanshen. The purpose of this study was to develop an HPLC-UV method for simultaneous determination of harpagoside and cinnamic acid in rat plasma and investigate pharmacokinetic parameters of harpagoside and cinnamic acid after oral administration of xuanshen extract (760 mg kg(-1)). After addition of syringin as internal standard, the analytes were isolated from plasma by liquid-liquid extraction. Separation was achieved on a Kromasil C18 column, and detection was by UV absorption at 272 nm. The described assay was validated in terms of linearity, accuracy, precision, recovery, and limit of quantification according to the FDA validation guidelines. Calibration curves for both analytes were linear with the coefficient of variation (r) for both was greater than 0.999. Accuracy for harpagoside and cinnamic acid ranged from 100.7-103.5% and 96.9-102.9%, respectively, and precision for both analytes were less than 8.5%. The main pharmacokinetic parameters found for harpagoside and cinnamic acid after oral infusion of xuanshen extract were as follows: Cmax 1488.7 +/- 205.9 and 556.8 +/- 94.2 ng mL(-1), Tmax 2.09 +/- 0.31 and (1.48 +/- 0.14 h, AUC(0-24) 10,336.4 +/- 1426.8 and 3653.1 +/- 456.4 ng h mL(-1), AUC(0-infinity) 11,276.8 +/- 1321.4 and 3704.5 +/- 398.8 ng h mL(-1), and t(1/2) 4.9 +/- 1.3 and 2.5 +/- 0.9 h, respectively. These results indicated that the proposed method is simple, selective, and feasible for pharmacokinetic study of radix Scrophulariae extract in rats.

HPLC method for the determination and pharmacokinetic studies on geniposide in rat serum after oral administration of traditional Chinese medicinal preparation Yin-Zhi-Ku decoction.

A new HPLC method for the determination of geniposide in rat serum with solid-phase extraction (SPE) for preconcentration is described. Geniposide and an internal standard (paeoniflorin) were extracted from serum by SPE using C18 cartridges. Analysis of the extract was then performed on a reversed-phase C18 column using acetonitrile-water (16:84, v/v) as the eluting solvent system, and UV detection at 238 nm was used to measure the analyte with a limit of quantitation about 0.1 microg/mL. The calibration curve for geniposide was linear (r = 0.9993) in the concentration range 0.1-16.0 microg/mL. The intra- and inter-day precision of the geniposide were determined and their RSD did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of geniposide from rat serum after oral administration of Yin-Zhi-Ku decoction.

Immunosuppressive iridoids from the fruits of Gardenia jasminoides.

A new iridoid, gardaloside (1), and a new safranal-type monoterpene, jasminoside G (2), together with 10 known compounds including nine iridoids and a second safranal-type monoterpene, were isolated from the fruits of Gardenia jasminoides. The structures of 1 and 2 were established on the basis of spectroscopic evidence. Of these compounds, geniposide (3), 6alpha-hydroxygeniposide (5), ixoroside (7), and shanzhiside (8) showed significant inhibition of IL-2 secretion by phorbol myristate acetate and anti-CD28 monoclonal antibody co-stimulated activation of human peripheral blood T cells.

Investigations on the pharmacokinetic properties of Harpagophytum extracts and their effects on eicosanoid biosynthesis in vitro and ex vivo.

PURPOSE: Harpagophytum extract and its marker substance harpagoside were shown to exert anti- inflammatory effects by interacting with the eicosanoid biosynthesis. In this study, different Harphagophytum extracts were tested with respect to inhibition of leukotriene and thromboxane biosynthesis in vitro and ex vivo. In addition, pharmacokinetic parameters of Harpagophytum extracts were investigated in vivo. METHODS AND SUBJECTS: Different fractions of Harpagophytum extracts were tested in vitro in human whole blood samples for effects on basal and ionophore A23187-stimulated cysteinyl-leukotriene (Cys-LT) and thromboxane synthesis. Furthermore, in 3 independent studies with different numbers of human male volunteers, a Harpagophytum extract was administered orally and tested in whole blood samples for Cys-LT and thromboxane B2 (TXB2) biosynthesis and for the determination of pharmacokinetic parameters of harpagoside. RESULTS: The special Harpagophytum extract WS1531 had a stronger inhibitory effect on ionophore A23187-stimulated Cys-LT levels compared with pure harpagoside or other extract fractions. Fractions without harpagoside had no pronounced inhibitory effect. When Cys-LT levels were measured after oral intake of Harpagophytum extract, a biphasic but dose-independent decrease of 28% and 58%, respectively, in basal Cys-LT formation was observed. Pharmacokinetic studies with the Harpagophytum extract WS1531 showed that the maximum levels of plasma harpagoside were reached after 1.3 to 2.5 hours. A linear relationship between dose and the first maximal concentration (Cmax) or area under the curve (AUC) (0-1)/AUC(0-infinity) was observed. CONCLUSIONS: Our observations strongly indicate a close relation between serum harpagoside levels and the inhibition of leukotriene biosynthesis.

[Studies on the metabolism of gentiopicroside by rat intestinal flora].

OBJECTIVE: To study the metabolism of gentiopicroside by rat intestinal flora in vitro and time profiles of gentiopicroside and its metabolites in rat serum after oral administration of gentiopicroside. METHOD: Using high performance liquid chromatography (HPLC) for determination of gentiopicroside, gentianine and gentianal content of rat intestinal flora culture medium in vitro and rat serum sample in vivo. RESULT: Gentiopicroside was metabolized quickly in vitro by rat intestinal flora, consequently gentianine and gentianal content appeared. Gentianine and gentianal were observed after gentiopicroside oral administration 4 times in rat serum sample, and gentiopicroside content decreased after oral administration 6 times. CONCLUSION: Gentianine and gentianal were the important bioactive metabolites of gentiopicroside in rats.