Determination of S-propargyl-cysteine in rat plasma by mixed-mode reversed-phase and cation-exchange HPLC-MS/MS method and its application to pharmacokinetic studies.

A simple, fast and sensitive mixed-mode reversed-phase and cation-exchange HPLC-MS/MS method for the quantification of S-propargyl-cysteine (SPRC), a novel cardioprotective agent, has been developed and validated for preclinical studies. Chromatographic separation of SPRC and its internal standard (IS) was performed using a commercial analytical column which contained both C18 bonded silica particles and sulfonic acid cation-exchange particles. The optimized mobile phase was composed of acetonitrile/ammonium acetate buffer (10mM, pH 4): 30/70 (v/v). Quantification was conducted by multiple reaction monitoring (MRM) of the transitions of m/z 160.0 → 143.0 for SPRC and 178.1 → 160.9 for S-butyl-cysteine (IS). The assay utilized methanol to achieve a simple and fast deproteinization. The lower limit of quantification (LLOQ) was 0.6 µg/mL (diluted with 50-fold of methanol) using 20 µL rat plasma. The assay was linear over a range from 0.6 to 159 µg/mL, with intra- and inter-batch accuracy (as relative error) less than ± 5% and precision (as relative standard deviation) less than 10%. Using the validated assay, the pharmacokinetic properties of SPRC in rats were investigated. SPRC exhibits linear pharmacokinetics after oral or intravenous administration in rats. The bioavailability after oral administration at 25, 75, and 225 mg/kg was 96.6%, 97.0%, and 94.7%, respectively.

20(S)-ginsenoside Rh2 noncompetitively inhibits P-glycoprotein in vitro and in vivo: a case for herb-drug interactions.

P-glycoprotein (P-gp) is an ATP-dependent efflux transporter highly expressed in gastrointestinal tract and multidrug resistance tumor cells. Inhibition or induction of P-gp can cause drug-drug interactions and thus influence the effects of P-gp substrate drugs. Previous studies indicated that 20(S)-ginsenoside Rh2 [20(S)-Rh2] could synergistically enhance the anticancer effects of conventional chemotherapeutic agents at a nontoxic dose. The aim of present study was to investigate in vitro and in vivo whether 20(S)-Rh2 was a P-gp inhibitor and analyze the possible inhibitory mechanisms and potential herb-drug interactions. Results showed that in vitro, 20(S)-Rh2 significantly enhanced rhodamine 123 retention in cells and decreased the efflux ratio of digoxin, fexofenadine, and etoposide, which were comparable to the effects of the established P-gp inhibitor verapamil. However, the transport of 20(S)-Rh2 suggested that 20(S)-Rh2 was not a P-gp substrate. Furthermore, the inhibitory effect persisted for at least 3 h after removal of 20(S)-Rh2. Unlike P-gp substrates, 20(S)-Rh2 inhibited both basal and verapamil-stimulated P-gp ATPase activities. It also significantly decreased UIC2 binding fluorescence, a marker for conformational change of P-gp. In situ and in vivo experiments showed that 20(S)-Rh2 increased the area under the plasma concentration-time curve and maximum plasma concentration of digoxin, fexofenadine, and etoposide significantly without affecting terminal elimination half-time. Long-term treatment with 20(S)-Rh2 failed to affect intestinal P-gp expression in vitro and in vivo. In conclusion, 20(S)-Rh2 is a potent noncompetitive P-gp inhibitor, which indicates a potential herb-drug interaction when 20(S)-Rh2 is coadministered with P-gp substrate drugs. It could increase the absorption of P-gp substrate drugs without long-term induction of P-gp expression in rats.

23-Hydroxybetulinic acid from Pulsatilla chinensis (Bunge) Regel synergizes the antitumor activities of doxorubicin in vitro and in vivo.

UNLABELLED: ETHNOPHARMACOLOGICAL REVELANCE: Pulsatilla chinensis (Bunge)Regel has been used as adjuvant in chemotherapy in traditional Chinese medicine. 23-Hydroxybetulinic acid, an isolated pentacyclic triterpene, is the major active constituent of Pulsatilla chinensis (Bunge) Regel. AIM OF THIS STUDY: To evaluate the combinational anticancer effect of 23-hydroxybetulinic acid and doxorubicin in vitro and in vivo. MATERIALS AND METHODS: The effect of combination treatment with 23-hydroxybetulinic acid and doxorubicin was evaluated with a quantitative combination index method based on the median-effect analysis in various cancer cell lines. And in vivo efficacy of combination chemotherapy was also evaluated using ICR mice bearing sarcoma 180 carcinoma tumors. RESULTS: 23-Hydroxybetulinic acid showed a synergistic cytotoxic effect on multiple cancer cell lines by combined use with doxorubicin. In vivo studies further demonstrated that co-administration of 23-HBA significantly improved the sensitivity of the tumor to doxorubicin through increasing intra-tumor doxorubicin concentration and inhibiting doxorubicin-induced up-regulation of P-gp in tumor. CONCLUSION: These results suggest that the combined therapy with 23-hydroxybetulinic acid and doxorubicin may be a new promising strategy to promote the clinical chemotherapy, which needs further verification.

Metabonomic phenotype and identification of "heart blood stasis obstruction pattern" and "qi and yin deficiency pattern" of myocardial ischemia rat models.

The traditional Chinese medicine concepts of "Xinxueyuzuzheng (heart blood stasis obstruction pattern)" and "Qiyinliangxuzheng (qi and yin deficiency pattern)" for myocardial ischemia rat models were constructed in the present study. Endogenous metabolites in rat plasma were analyzed using the GC/TOF-MS-based metabonomic method. Significant metabolic differences were observed between the control and two model groups, and the three groups were distinguished clearly by pattern recognition. Compared with those of the control, the levels of hydroxyproline, threonic acid, glutamine and citric acid were strikingly up- or down-regulated in model rats. The metabolites contributing most to the classification between the two "pattern" rats were identified, such as valine, serine, threonine, ornithine, hydroxyproline, lysine, 2-hydroxybutanoic acid, 3-hydroxybutanoic acid, galactofuranose and inositol. These compounds were indicated as the potential biomarkers. The results suggested that the two "patterns" are involved in dysfunction in oxidative stress, energy metabolism and amino acid metabolism. These findings also provided the substantial foundation for exploring the scientific connotation of these two "Zhengxing (pattern types)" of myocardial ischemia, and "Bianzheng (pattern identification)".

Pharmacokinetic characterization of ginsenoside Rh2, an anticancer nutrient from ginseng, in rats and dogs.

The pharmacokinetic characteristics of ginsenoside Rh2, an anticancer nutrient, were analyzed in dogs and rats, including plasma kinetics, bioavailability, tissue distribution, plasma protein binding and excretion. The bioavailability of Rh2 is about 5% in rats and 16% in dogs. Multiple-dosing (7 days, 1 mg/kg bid) did not affect the pharmacokinetics in dogs. After oral dosing, Rh2 distributed mainly to the liver and gastrointestinal tissues in rats. In rats, the circulating fraction of Rh2 bound to plasma proteins was around 70%. The systemic clearance, however, was low -- around 2 and 20 ml/min/kg in dogs and rats, respectively. Only 1% of dosed Rh2 were recovered in excreta of rats as the intact form after oral administration, while 30% was excreted unchanged in bile after i.v. dosing. We subsequently investigated the membrane permeability of Rh2 across Caco-2 cell monolayers, stability and elimination profiles in the gastrointestinal environment. Low membrane permeability (P(app)(AP-BL): 1.91 x 10(-8)cm/s), efflux transport (efflux ratio: 9.8), pre-systemic elimination (degradation in acidic condition; metabolism in intestine tissue and contents), as well as low solubility largely accounted for the low bioavailability of Rh2. Regarding the low solubility of Rh2, micronization of the dose almost doubled the rate of absorption in dogs. Preliminary metabolite profiling confirmed the presence of the deglycosidating product protopanaxadiol in rat feces. A possible metabolite in rat bile and a potential sulfate-conjugate in rat urine were also detected.

Construction of the fingerprints of ginseng stem and leaf saponin reference substances and spiked plasma sample by LC-ESI/MS and its application to analyzing the compounds absorbed into blood after oral administration of ginseng stem and leaf saponin in rat.

Develop a simple and reliable assay method to detect ginseng stem and leaf saponins (GSLS) in methanol and rat plasma by liquid chromatography-electrospray ionization mass spectrometry in scan mode, and construct the fingerprints of GSLS reference substances and plasma samples. In order to screen the active constituents of GSLS, analysis and comparison were carried out between the LC/ESI-MS profiles of blank rat plasma and rat plasma samples obtained after oral administration of GSLS. Thirty-one compounds were detected and 10 of them were identified in the fingerprints of reference substances and spiked plasma sample. Furthermore, 12 compounds (C7, C8, C14, C15, C18, Re, C24, Rb(1), Rc, Rb(2), Rb(3), Rd) were absorbed easily and some new compounds were generated after oral administration of GSLS, which might be the metabolites of GSLS. These absorbed components and new compounds may be the main bioactive components of GSLS.

Pharmacokinetic and absolute bioavailability study of total panax notoginsenoside, a typical multiple constituent traditional chinese medicine (TCM) in rats.

LC/ESI/MS method was employed for the pharmacokinetic evaluation of total panax notoginsenoside (TPNS) in rats. After oral or intravenous administration of TPNS at the dosage of 300.0 or 10.0 mg kg(-1) to rats respectively, panax notoginsenoside R1, ginsenoside Rg1, Rd, Re and Rb1 were simultaneous determined in rat plasma. Pharmacokinetic parameters and absolute bioavailability of panax notoginsenoside R1, ginsenoside Rg1, Rd, Re and Rb1 were obtained by the Drug And Statistics for windows (DAS) pharmacokinetic software. The pharmacokinetic parameters of all analytes were different form each other. T(1/2) were changed from 0.72 to 22.16 h and AUC were changed from 1.03 to 98.94 mg/l.h after oral or intravenous administration TPNS or Xuesaitong (TPNS) injection. The absolute bioavailability of R1, Rg1, Rd, Re and Rb1 were of 9.29%, 6.06%, 2.36%, 7.06% and 1.18%, respectively.

Simultaneous determination of panax notoginsenoside R1, ginsenoside Rg1, Rd, Re and Rb1 in rat plasma by HPLC/ESI/MS: platform for the pharmacokinetic evaluation of total panax notoginsenoside, a typical kind of multiple constituent traditional Chinese medicine.

A platform for the pharmacokinetic study of multiple constituent traditional Chinese medicine was developed and validated. An HPLC/ESI/MS method was employed for the simultaneous determination of panax notoginsenoside R1, ginsenoside Rg1, Rd, Re and Rb1 in rat plasma. After the addition of digoxin as an internal standard (IS), rat plasmas were extracted with n-butanol saturated with pure water and all analytes were separated on a reversed-phased C(18) column with a mobile phase of acetonitrile-water (0.5 mM ammonium chloride) and pumped at a flow rate of 0.2 mL/min. Analytes were determined in a single quadrupole mass spectrometer using an electrospray ionization source. HPLC/ESI/MS was performed in the selected-ion monitoring mode with the chlorinated adducts of molecular ions [M + Cl]( -) at m/z 967.75, 835.80, 981.80, 981.80, 1143.65 and 815.40 for R1, Rg1, Rd, Re, Rb1 and digoxin, respectively. The method showed excellent linearity over the concentration range 3.03-775.00 ng/mL (r(2) = 0.9994) for R1, 4.00-1025.00 ng/mL (r(2) = 0.9991, 0.9988, 0.9991) for Rg1, Rd and Re, respectively, and 2.77-710.00 ng/mL for Rb1 (r(2) = 0.9990). The low limit of quantification was 3.03, 4.00, 4.00, 4.00 and 2.77 ng/mL for R1, Rg1, Rd, Re and Rb1, respectively, with S/N > 10. The intra- and inter-day precisions were below 12.00% and the accuracy was between -2.31 and +4.43% for all analytes. The extract recoveries of analytes were from 67.47 to 94.18%. All analytes were stable in rat plasma after storage for 12 h at ambient temperature, at 4 degrees C for 12 h in the sample pool, at -20 degrees C for 4 weeks and at -20 degrees C for three thaw-freeze cycles. The HPLC/ESI/MS technique provided an excellent method for the simultaneous quantification of R1, Rg1, Rd, Re and Rb1 in rat plasma and was successfully applied to the pharmacokinetic study of a multiple-constituent traditional Chinese medicine, total panax notoginsenoside (Xuesaitong injection).