Mechanism-Based Pharmacokinetic Model for the Deglycosylation Kinetics of 20(S)-Ginsenosides Rh2.

Aim: The 20(S)-ginsenoside Rh2 (Rh2) is being developed as a new antitumor drug. However, to date, little is known about the kinetics of its deglycosylation metabolite (protopanoxadiol) (PPD) following Rh2 administration. The aim of this work was to 1) simultaneously characterise the pharmacokinetics of Rh2 and PPD following intravenous and oral Rh2 administration, 2) develop and validate a mechanism-based pharmacokinetic model to describe the deglycosylation kinetics and 3) predict the percentage of Rh2 entering the systemic circulation in PPD form. Methods: Plasma samples were collected from rats after the I.V. or P.O. administration of Rh2. The plasma Rh2 and PPD concentrations were determined using HPLC-MS. The transformation from Rh2 to PPD, its absorption, and elimination were integrated into the mechanism based pharmacokinetic model to describe the pharmacokinetics of Rh2 and PPD simultaneously at 10 mg/kg. The concentration data collected following a 20 mg/kg dose of Rh2 was used for model validation. Results: Following Rh2 administration, PPD exhibited high exposure and atypical double peaks. The model described the abnormal kinetics well and was further validated using external data. A total of 11% of the administered Rh2 was predicted to be transformed into PPD and enter the systemic circulation after I.V. administration, and a total of 20% of Rh2 was predicted to be absorbed into the systemic circulation in PPD form after P.O. administration of Rh2. Conclusion: The developed model provides a useful tool to quantitatively study the deglycosylation kinetics of Rh2 and thus, provides a valuable resource for future pharmacokinetic studies of glycosides with similar deglycosylation metabolism.

Pharmacokinetics of the prototype and hydrolyzed carboxylic forms of ginkgolides A, B, and K administered as a ginkgo diterpene lactones meglumine injection in beagle dogs.

Ginkgo diterpene lactones meglumine injection (GDLI) is a commercially available product used for neuroprotection. However, the pharmacokinetic properties of the prototypes and hydrolyzed carboxylic forms of the primary components in GDLI, i.e., ginkgolide A (GA), ginkgolide B (GB), and ginkgolide K (GK), have never been fully evaluated in beagle dogs. In this work, a simple, sensitive, and reliable method based on ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was developed, and the prototypes and total amounts of GA, GB, and GK were determined in beagle dog plasma. The plasma concentrations of the hydrolyzed carboxylic forms were calculated by subtracting the prototype concentrations from the total lactone concentrations. For the first time, the pharmacokinetics of GA, GB, and GK were fully assessed in three forms, i.e., the prototypes, the hydrolyzed carboxylic forms, and the total amounts, after intravenous administration of GDLI in beagle dogs. It was shown that ginkgolides primarily existed in the hydrolyzed form in plasma, and the ratio of hydrolysates to prototype forms of GA and GB decreased gradually to a homeostatic ratio. All of the three forms of the three ginkgolides showed linear exposure of AUC to the dosages. GA, GB, and GK showed a constant half-life approximately 2.7, 3.4, and 1.2 h, respectively, which were consistent for the forms at three dose levels (0.3, 1.0, and 3.0 mg·kg-1) and after a consecutive injection of GDLI for 7 days (1.0 mg·kg-1).

[Metabolomic approach to evaluating the effect of the mixed decoction of kelp and licorice on system metabolism of SD rats].

The aim of the study is to evaluate the effects of the single and mixed decoction of Thallus laminariae (kelp) and Glycyrrhiza glabra (licorice) on the metabolism and their difference. The mixed decoction of kelp and licorice and the single decoction were made and intragastrically administered to the SD rats. The effect on system metabolism, the toxicity of liver and kidney were assessed by GC-MS profiling of the endogenous molecules in serum, routine biochemical assays and histographic inspection of tissues from SD rats, separately. The mixed decoction of kelp and licorice induced more obvious pathological abnormalities in SD rats than a single decoction of kelp, while the extracts of licorice did not show any pathological change. Neither the mixed, nor the single decoction showed abnormal histopathology. After intragastric administration of extracts for 5 days, the mixed decoction induced a decrease of ALT (no significant change in the groups of single decoction) and an increase of BUN (so did the single decoction of kelp). Metabolomic profile of the molecules in serum revealed that the metabolic patterns were all obviously affected for the three groups, i.e., the mixed and single decoction of kelp and licorice. The rats given with the single decoction of kelp showed a similar pattern to that of the mixed decoction, indicating that the kelp primarily contributed the perturbation of metabolism for the mixed decoction. All three groups induced a decrease of branched chain amino acids, TCA cycle intermediates and glycolysis intermediates (e.g., pyruvic acid and lactic acid) and an increase of 3-hydroxybutyric acid. Kelp decoction showed stronger potential in reducing TCA cycle intermediates and glycolysis intermediates than the other two groups, while the levels of branched chain amino acids were the lowest after licorice extracts were given. These results suggested that the effect of the mixed decoction on metabolism was closely associated with both kelp and licorice. The continuous administration of single decoction of kelp and the mixed decoction of licorice and kelp resulted in pathological abnormalities in kidney of SD rats. The mixed decoction of kelp and licorice distinctly perturbed sera molecules and hence system metabolism, which showed associated with those of kelp and licorice. Although the metabolic effect was associated with both kelp and licorice, the results suggested kelp contributed to it primarily.

[Plasma metabonomics study of ischemic cerebral apoplexy rats treated with Tongsaimai pellets].

OBJECTIVE: To observe abnormal metabolic changes caused by ischemic cerebral apoplexy and the regulating action of Tongsaimai pellets on abnormal metabolism by analyzing the change of small molecules in plasma of ischemic cerebral apoplexy rat. To find the potential biomarkers, and to explore metabolic mechanisms of Tongsaimai pellets. METHOD: Rat models of middle cerebral artery occlusion was established with electric coagulation, and rats were divided into 4 groups, model group, sham-operation group, Tongsaimai pellets group and positive control group. Tongsaimai pellets and positive control group were orally administrated by 13.2 g x kg(-1) x d(-1) of crude drugs and 32 mg x kg(-1) x d(-1) of Nimodipine respectively, m odel and sham-operation group by equal volume of distilled water for a week. Plasma of model and sham-operation group were collected, and plasma of Tongsaimai pellets and positive control group were collected on the 1st, 3rd , 7th day after administration. Endogenous metabolites of four groups were determined with GC-MS. Partial least squares discriminant analysis (PLS-DA) was applied to analyze multivariate data and set up model, and T-test was used in significant statistical analysis. RESULT: Compared with sham-operation group rats, pyruvic acid, taurine and hydroxyproline obviously increased in model group rats, while lactic acid, glyceric acid, aminomalonic acid, fructose, tryptophan and leucine significantly decreased, so these metabolites were potential metabolic biomarkers. These endogenous metabolites except taurine got restoration in Tongsaimai group rats. CONCLUSION: Abnormal metabolite level in plasma can be certainly recovered by Tongsaimai pellets, and the treatment of Tongsaimai pellets can be connected with the regulation of related metabolic pathways.

Rapid identification of ophiopogonins and ophiopogonones in Ophiopogon japonicus extract with a practical technique of mass defect filtering based on high resolution mass spectrometry.

This study was to develop and evaluate a practical approach of mass defect filtering (MDF), a post-acquisition data processing technique, for the rapid classification of complicated peaks into well-known chemical families based on the exact mass acquired by high resolution mass spectrometry. The full-scan LC-MS/MS data of the Ophiopogon japonicus extract was acquired using high performance liquid chromatography coupled with hybrid quadrupole-time of flight (LCMS-Q-TOF) system which features high resolution, mass accuracy, and sensitivity. To remove the interferences of the complex matrix, MDF approach was developed and employed to rapidly pick out the peaks of ophiopogonins and ophiopogonones from full-scan mass chromatograms. The accuracy of MDF was evaluated in reference to the result of structural identification. After the MDF based classification, both target and non-target components in Ophiopogon japonicus extract were characterized based on the detailed fragment ions analysis in the hybrid ion trap and time-of-flight mass spectrometry (LCMS-IT-TOF). By this approach, more than 50 ophiopogonins and 27 ophiopogonones were structurally characterized. The present results of rapid detection and identification of ophiopogonins and ophiopogonones suggest that the proposed MDF approach based on the high-resolution mass spectrometry data would be expected adaptable to the analysis of other herbal components.

[Metabonomic phenotype of "formula corresponding to pattern types" based on "qi and yin deficiency pattern" of myocardial ischemia rat model].

In order to explore the scientific connotation of "Fangzhengduiying (formula corresponding to pattern types)", "Qiyinliangxuzheng (Qi and Yin deficiency pattern)" of myocardial ischemia rat model and GC-TOF/MS based metabonomic method were used for comparing the effects of Sheng-mai injection, Salvia injection and propranolol in the present study. After data processing and pattern recognition, Sheng-mai injection showed better efficacy than the other two drugs in accordance with not only visual observation from PLS-DA scores plots but also the number of abnormal endogenous compounds restored to the normal level. Further studies showed that Sheng-mai injection could normalize the level of plasma endothelin-1, the index related to cardiovascular diseases and sleep disorders, which verified the results of metabonomics. Finally, the regulated metabolites and related metabolic pathways were analyzed, and it was supposed that the effects of Sheng-mai injection involved in the alternation of energy metabolism, lipid metabolism, amino acids metabolism, and so on. These findings provided scientific evidence to Shengmai "Fang" used for "Qi and Yin deficiency pattern" correspondingly, indicating that metabonomics has great potential in traditional Chinese medical research, which provides a novel approach and way to modernization of traditional Chinese medicine.

Induction of cytochromes P450 1A1 and 1A2 by tanshinones in human HepG2 hepatoma cell line.

Diterpenoid tanshinones including tanshinone IIA (TIIA), cryptotanshinone (CTS), tanshinone I (TI) and dihydrotanshinone I (DHTI) are the major bioactive components from Danshen. The major aim of our present study was to investigate the induction potential of these four main components of tanshinones (TIIA, CTS, TI, and DHTI) on the expression of CYP1A1 and CYP1A2 in HepG2 cells. Our results showed that all of these four tanshinones caused a significant time- and concentration-dependent increase in the amount of CYP1A1/2 expression in HepG2 cells. These induction effects were further characterized through transcriptional regulation: the induction of CYP1A1/2 mRNA level by tanshinones was completely blocked by the transcription inhibitor actinomycin D; the expression of CYP1A1/2 heterogeneous nuclear RNA was induced by tanshinone treatment; and CYP1A1 mRNA stability was not influenced by these tanshinones. Interestingly, tanshinones plus B[a]P produced additive/synergistic effect on CYP1A1/2 induction. In addition, the tanshinone-induced CYP1A1/2 expression was abolished by the aryl hydrocarbon receptor (AhR) antagonist resveratrol, suggesting an AhR dependent transcription mechanism. In the reporter gene assay, while TI and DHTI significantly induced AhR-dependent luciferase activity, TIIA and CTS failed to induce this activity. Collectively, the tanshinones could induce CYP1A1 and CYP1A2 expression through transcriptional activation mechanism and exert differential effects on activating AhR in HepG2 cells. Our findings suggest that rational administration of tanshinones should be considered with respect to their effect on AhR and CYP1A1/2 expression.

Influence of segmental and selected ion monitoring on quantitation of multi-component using high-pressure liquid chromatography-quadrupole mass spectrometry: Simultaneous detection of 16 saponins in rat plasma as a case.

Quadrupole (Q) mass spectrometers are the most popular analytical tools due to their reliability, effectiveness, and low cost. However, they are not suitable for quantitative analysis of multi-component since the sensitivity will get worse rapidly with the increasing number of m/z detected. The present work, for the first time, attempted to analyze of 16 saponins simultaneously using an approach of segmental and selected ion monitoring (SSIM) based on LC-Q/MS, and systematically investigated the influence of different SSIM modes on signal level/noise level (S/N), lower limits of quantification (LLOQ), upper limits of quantification (ULOQs), etc. Our results showed that a proper SSIM mode could not only provide much higher sensitivity for all the targeting analytes, but also dramatically broadened their dynamic ranges. The developed methodology could effectively break the application bottleneck on the quantitative analysis of multi-component with LC-Q/MS, and would be applied widely in related fields for multi-component analysis, such as environmental monitoring, metabonomics, Chinese herbal medicine research.

Application of GC/MS-based metabonomic profiling in studying the lipid-regulating effects of Ginkgo biloba extract on diet-induced hyperlipidemia in rats.

AIM: To evaluate the lipid-regulating effects of extract from Ginkgo biloba leaves (EGB) using pharmacological methods and metabonomic profiling in a rat model of diet-induced hyperlipidemia. METHODS: EGB was orally administered at a dose level of 40 mg/kg in both the EGB-prevention and -treatment groups. All rat samples obtained were examined for known and potential biomarkers and enzyme activity using commercial assay kits and GC/MS-based metabonomic profiling coupled with principal component analysis (PCA). RESULTS: The data obtained from the assay kits indicated that EGB reduced total cholesterol and low density lipoprotein cholesterol levels and increased high density lipoprotein cholesterol levels in rat plasma obtained from both the EGB-prevention and -treatment groups compared with those of the diet-induced hyperlipidemia group. EGB also increased the activities of lipoprotein lipase and hepatic lipase and excretion of fecal bile acid in rats from the EGB-prevention and-treatment groups. Using GC/MS-based metabonomic analysis, more than 40 endogenous metabolites were identified in rat plasma. PCA of rat plasma samples obtained using GC/MS produced a distinctive separation of the four treatment groups and sampling points within each group. Metabolic changes during hyperlipidemia formation and improvement resulting from EGB treatment were definitively monitored with PCA score plots. Furthermore, elevated levels of sorbitol, tyrosine, glutamine and glucose, and decreased levels of citric acid, galactose, palmitic acid, arachidonic acid, acetic acid, cholesterol, butyrate, creatinine, linoleate, ornithine and proline, were observed in the plasma of rats treated with EGB. CONCLUSION: EGB exerts multi-directional lipid-lowering effects on the rat metabonome, including limitation of the absorption of cholesterol, inactivation of HMGCoA and favorable regulation of profiles of essential polyunsaturated fatty acid (EFA). Further experiments are warranted to explore the mechanisms of action underlying the lipid-regulating effects of EGB against hyperlipidemia.

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)".

Study on LC-MS fingerprint for quality assessment of Aristolochia manshuriensis with chemical pattern recognition / 中国中药杂志

<p><b>OBJECTIVE</b>To analyze LC-MS fingerprints of Aristolochia manshuriensis for quality assessment with two different chemical pattern recognition models.</p><p><b>METHOD</b>LC-MS fingerprints of A. manshuriensis were established from 24 batches of samples from different habitats. SIMCA and Clustering analysis were used to compare the parameters of the 29 common peaks.</p><p><b>RESULT</b>Two methods had good consistency, while they reflected the inherent sample information from different perspectives, respectively.</p><p><b>CONCLUSION</b>Modern equipment analysis technology and multivariable chemical pattern recognition would be an efficient way for quality control and variety identification of A. manshuriensis.</p>

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 study of triptolide, a constituent of immunosuppressive chinese herb medicine, in rats.

Triptolide is a potential anti-immune agent, and has shown multi-organic toxicity, however its toxic mechanism remained undiscovered. This paper aimed at characterizing the pharmacokinetic profiles of triptolide in rats to provide the clue to approach the toxic mechanism. The absorption, distribution, metabolism and excretion of triptolide were investigated in male Sprague-Dawley rats after single doses of oral and i.v. administration. After oral administration of 0.6, 1.2 and 2.4 mg/kg, the concentration of triptolide in plasma reached the maximum within 15 min, and declined rapidly with an elimination half-life from 16.81 to 21.70 min. The triptolide kinetics was fitted into one-compartment model after i.v. administration. Oral absolute bioavailability was 72.08% at the dose of 0.6 mg/kg. Triptolide was also rapidly distributed and eliminated in all selected tissues. Less than 1% triptolide of the dose was recovered from the bile, urine or feces as parent drug within 48 h. While triptolide could not be detected in tissues and plasma at 4 h post dose, rats in the group C (oral: 1.2 mg/kg) and D (oral: 2.4 mg/kg) showed obvious toxic response to triptolide and some of rats even died out. It was indicated that triptolide was metabolized extensively, eliminated rapidly, and also showed that the toxicity produced by the triptolide was lag behind the exposure concentration.

Transport and bioavailability studies of astragaloside IV, an active ingredient in Radix Astragali.

Astragaloside IV is an important constituent of Radix Astragali, a herbal remedy widely used in traditional Chinese medicine. Radix Astragali is administered orally but little is known about the transport properties and bioavailability of astragaloside IV. In this paper we report studies of the absorption of astragaloside IV in the perfused rat intestinal model, transport and uptake in Caco-2 cell monolayers and in vivo bioavailability in rat after an oral dose. In the perfused rat intestinal model, absorption of astragaloside IV was low from an aqueous solution but was significantly higher from a solution of Radix Astragali. Absorption was not affected by bile duct ligation. Transport through Caco-2 cells gave a very low permeability value (mean P(app) of 6.7+/-1.0 x 10(-8) cm/sec.) and uptake was unaffected by P-glycoprotein inhibitors. The absolute bioavailability of astragaloside IV in rat was 2.2%. The correlation between low permeability in vitro and poor bioavailability in vivo indicates in vitro absorption studies are useful in the evaluation of traditional Chinese medicines.

[Study on the metabolites of guanfu base A hydrochloride in rat urine by high performance liquid chromatograph-mass spectrum].

AIM: To study the metabolites of guanfu base A hydrochloride (GFA) in rat urine. METHODS: Rat urine was collected after i.v. injection of GFA. Phase I metabolites were identified by HPLC/MS and by comparison with authentic standards. Phase II conjugates were treated with either glucuronidase or sulfatase in presence or absence of glucuronidase specific inhibitor D-saccharic acid beta-1,4-lactone. The aglycones were identified by HPLC/MS. RESULTS: Phase I metabolites guanfu base I (GFI) and guanfu alcohol-amine (AA) were separated and identified in rat urine by comparison with authentic standards. Phase II conjugates, for which no authentic standards were available, GFA glucuronide and sulfate conjugates, GFI glucuronide and sulfate were separated and tentatively identified by hydrolysis with glucuronidase or sulfatase, the aglycones, GFA and GFI, were identified in rat urine. CONCLUSION: After i.v. injection of GFA, GFA is metabolized into GFI, AA, GFA glucuronide and sulfate conjugates, GFI glucuronide and sulfate conjugates in rat urine. The polarity of the metabolites is increased, and the effectiveness of them is lower than the parent drug.