Oleuropein-Induced Acceleration of Cytochrome P450-Catalyzed Drug Metabolism: Central Role for Nuclear Receptor Peroxisome Proliferator-Activated Receptor α.

Oleuropein (OLE), the main constituent of Olea europaea, displays pleiotropic beneficial effects in health and disease, which are mainly attributed to its anti-inflammatory and cardioprotective properties. Several food supplements and herbal medicines contain OLE and are available without a prescription. This study investigated the effects of OLE on the main cytochrome P450s (P450s) catalyzing the metabolism of many prescribed drugs. Emphasis was given to the role of peroxisome proliferator-activated receptor α (PPARα), a nuclear transcription factor regulating numerous genes including P450s. 129/Sv wild-type and Ppara-null mice were treated with OLE for 6 weeks. OLE induced Cyp1a1, Cyp1a2, Cyp1b1, Cyp3a14, Cyp3a25, Cyp2c29, Cyp2c44, Cyp2d22, and Cyp2e1 mRNAs in liver of wild-type mice, whereas no similar effects were observed in Ppara-null mice, indicating that the OLE-induced effect on these P450s is mediated by PPARα. Activation of the pathways related to phosphoinositide 3-kinase/protein kinase B (AKT)/forkhead box protein O1, c-Jun N-terminal kinase, AKT/p70, and extracellular signal-regulated kinase participates in P450 induction by OLE. These data indicate that consumption of herbal medicines and food supplements containing OLE could accelerate the metabolism of drug substrates of the above-mentioned P450s, thus reducing their efficacy and the outcome of pharmacotherapy. Therefore, OLE-induced activation of PPARα could modify the effects of drugs due to their increased metabolism and clearance, which should be taken into account when consuming OLE-containing products with certain drugs, in particular those of narrow therapeutic window. SIGNIFICANCE STATEMENT: This study indicated that oleuropein, which belongs to the main constituents of the leaves and olive drupes of Olea europaea, induces the synthesis of the major cytochrome P450s (P450s) metabolizing the majority of prescribed drugs via activation of peroxisome proliferator-activated receptor α. This effect could modify the pharmacokinetic profile of co-administered drug substrates of the P450s, thus altering their therapeutic efficacy and toxicity.

Detection and Identification of Catalpol Metabolites in the Rat Plasma, Urine and Faeces Using Ultra-high Performance Liquid Chromatography-Q Exactive Hybrid Quadrupole-orbitrap High-resolution Accurate Mass Spectrometry.

BACKGROUND: Catalpol, an iridoid glycoside, is one of the richest bioactive components present in Rehmannia glutinosa. More and more metabolites of drugs have exhibited various pharmacological effects, thus providing guidance for clinical application. However, few researches have paid attention to the metabolism of catalpol. OBJECTIVE: This study aimed to establish a rapid and effective method to identify catalpol metabolites and evaluate the biotransformation pathways of catalpol in rats. METHODS: In this study, catalpol metabolites in rat urine, plasma and faeces were analyzed by UHPLC-Q-Exactive MS for the characterization of the metabolism of catalpol. Based on high-resolution extracted ion chromatograms (HREICs) and parallel reaction monitoring mode (PRM), metabolites of catalpol were identified by comparing the diagnostic product ions (DPIs), chromatographic retention times, neutral loss fragments (NLFs) and accurate mass measurement with those of catalpol reference standard. RESULTS: A total of 29 catalpol metabolites were detected and identified in both negative and positive ion modes. Nine metabolic reactions, including deglycosylation, hydroxylation, dihydroxylation, hydrogenation, dehydrogenation, oxidation of methylene to ketone, glucuronidation, glycine conjugation and cysteine conjugation, were proposed. CONCLUSION: A rapid and effective method based on UHPLC-Q-Exactive MS was developed to mine the metabolism information of catalpol. Results of metabolites and biotransformation pathways of catalpol suggested that when orally administrated, catalpol was firstly metabolized into catalpol aglycone, after which phase I and phase II reactions occurred. However, hydrophilic chromatography-mass spectrometry is still needed to further find the polar metabolites of catalpol.

Determination of aucubin by supramolecular solvent-based dispersive liquid-liquid microextraction and UPLC-MS/MS: Application to a pharmacokinetic study in rats with type 1 diabetes.

A novel method was developed for the determination of aucubin in rat serum by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with supramolecular solvent (SUPRAS)-based dispersive liquid-liquid microextraction (DLLME). The SUPRAS was prepared with pentanol as extraction solvent and tetrahydrofuran as dispersing agent. Based on single factor test and response surface methodology, critical parameters were optimized as: 1 mL of pentanol as extraction solvent, 4 mL of tetrahydrofuran as dispersing agent, 200 µL of SUPRAS, and vortex duration of 2.5 min. The established method was validated in terms of selectivity, linearity, accuracy, precision, recovery, stability, and applied to a pharmacokinetic study on type 1 diabetes model rats intraperitoneally administered with aucubin. The experimental results showed that the maximum concentration in serum (Cmax) and area under the serum concentration versus time curve (AUC) of aucubin in type 1 diabetic rats were higher than those in normal rats. Such pharmacokinetic alterations might presumably result from the pathological state of type 1 diabetes. The current study may provide scientific evidence and inspiring insights for clinical application of aucubin for the treatment of diabetes.

Simultaneous determination of multiple components in rat plasma and pharmacokinetic studies at a pharmacodynamic dose of Xian-Ling-Gu-Bao capsule by UPLC-MS/MS.

Xian-Ling-Gu-Bao capsule (XLGB) is an effective traditional Chinese medicine prescription (TCMP) that is used for the prevention and treatment of osteoporosis in China. A rapid, simple, efficient and stable method based on UPLC-MS/MS technology was developed for simultaneous determination of multiple components of XLGB in rat plasma. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI). For twenty-one selected quantitative prototypes, all calibration curves showed favourable linearity (r>0.9932) in linear ranges. The lower limits of quantification (LLOQs) were 2 ng/mL for psoralen (PL), 2.5 ng/mL for asperosaponin VI (AS), 1 ng/mL for isopsoralen (IPS) and sweroside (SW), 0.5 ng/mL for magnoflorine (MA), bavachinin (BVN), tanshinone IIA (TA), timosaponin BII (TBII) and icaritin (ICT), 0.1 ng/mL for epimedin B (EB) and epimedin C (EC), 0.05 ng/mL for icariin (IC), isobavachalcone (IBC), psoralidin (PD), bavachin (BV), bavachalcone (BC), epimedin A (EA) and isobavachin (IBV), 0.02 ng/mL for neobavaisoflavone (NEO) and icariside I (ICI) and 0.01 ng/mL for icariside II (ICII). The intra-day and inter-day (low, medium, high) precision (relative standard deviation) for all analytes was less than 8.63%, and the accuracies (as relative error) were in the range of -12.45% to 8.91%. Extraction recoveries and matrix effects of analytes and IS were acceptable. All analytes were stable during the assay and storage in plasma samples. The validated method was successfully applied to the pharmacokinetics (PK) studies of the twenty-one prototypes at pharmacodynamic doses (0.3 and 1 g/kg/day). In addition, dynamic profiles of 28 metabolites (phase II conjugates: 23 glucuronide conjugates, 2 sulfate conjugates and 3 glucuronide or sulfate conjugates) were also monitored by their area/IS area-time curves. As a result, coumarins, prenylated flavonoids from Psoraleae Fructus, alkaloids and prenylated flavonol glycosides from Epimedii Herba, and iridoid glycosides, triterpenoid saponins from Dipsaci Asperoidis Radix were considered to be the key effective substances of XLGB due to their high exposure and appropriate pharmacokinetic features. This is the first report to reveal pharmacodynamic ingredients by a reversed pharmacodynamic (PD) - pharmacokinetics (PK) study.

Pharmacokinetics and metabolic profiles of swertiamarin in rats by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

Swertiamarin, a typical compound of secoiridiod glycosides with various pharmacological effects which is the major iridoid glicoside of Swertia. In this study, we have established a fast and sensitive LC-MS/MS method. The aim was to conduct pharmacokinetic studies of swertiamarin in vivo of rats. Gentiopicroside was used as internal standard and a C18 column was employed for the separation of analytes. The selected reaction monitoring transitions were m/z 375→177, 357.1→195 for swertiamarin and the internal standard, respectively, in a positive ion mode. The results showed that swertiamarin had a good linearity in the range of 2-8000 ng/mL (r ＞ 0.997) and its limit of detection (LLOD) was 0.5 ng/mL. The developed method subsequently successfully used in the pharmacokinetic study of swertiamarin in rats after oral administration (50, 100, and 150 mg/kg). We obtained a series of pharmacokinetic parameters, and the half-time of swertiamarin was 1 h, while the oral bioavailability was between 5.6-7.6%. Six metabolites of swertiamarin were identified based on accurate mass measurements of protonated molecules and their MS/MS spectrum by ultra-high-performance chromatography/tandem quadrupole time-of-flight mass spectrometry. Furthermore, metabolites were classified into three groups and the metabolic pathway of swertiamarin was proposed. The finding may help for the understanding of effectiveness and safety of swertiamarin.

Catalpol in Diabetes and its Complications: A Review of Pharmacology, Pharmacokinetics, and Safety.

This review aimed to provide a general view of catalpol in protection against diabetes and diabetic complications, as well as its pharmacokinetics and safety concerns. The following databases were consulted with the retrieval of more than 100 publications through June 2019: PubMed, Chinese National Knowledge Infrastructure, WanFang Data, and web of science. Catalpol exerts an anti-diabetic effect in different animal models with an oral dosage ranging from 2.5 to 200 mg/kg in rats and 10 to 200 mg/kg in mice. Besides, catalpol may prevent the development of diabetic complications in kidney, heart, central nervous system, and bone. The underlying mechanism may be associated with an inhibition of inflammation, oxidative stress, and apoptosis through modulation of various cellular signaling, such as AMPK/PI3K/Akt, PPAR/ACC, JNK/NF-κB, and AGE/RAGE/NOX4 signaling pathways, as well as PKCγ and Cav-1 expression. The pharmacokinetic profile reveals that catalpol could pass the blood-brain barrier and has a potential to be orally administrated. Taken together, catalpol is a well-tolerated natural compound with promising pharmacological actions in protection against diabetes and diabetic complications via multi-targets, offering a novel scaffold for the development of anti-diabetic drug candidate. Further prospective and well-designed clinical trials will shed light on the potential of clinical usage of catalpol.

Metabolic profiles and pharmacokinetics of picroside I in rats by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

Picroside I is an iridoid glycoside derived from Picrorhiza kurroa Royle ex Benth and Picrorhiza scrophulariiflora Pennell and characterized by many biological activities. In this study, a fast, selective, and sensitive UHPLC-MS/MS method was developed and validated to determine picroside I in rat plasma. Analytes were separated by using an ACQUITY UPLC® BEH C18 (2.1 × 50 mm, 1.7 µm) column at a running time of 2 min. Selected reaction monitoring (SRM) transitions were m/z 491.1 → 147.1 for picroside I and m/z 511.1 → 235.1 for the internal standard in a negative ion mode. The established UHPLC-MS/MS method achieved good linearity for picroside I within the range of 0.1-500 ng/mL. The validated method was successfully applied for the pharmacokinetic analysis of picroside I in rats after oral administration. Fifteen metabolites of picroside I were tentatively identified through ultra-high-performance chromatography/tandem quadrupole time-of-flight mass spectrometry, and four metabolites were identified by comparing with the standards. Besides, nine of these metabolites were discovered for the first time. The proposed metabolic pathways of picroside I in vivo can be divided into four parts, namely, phase I reaction of picroside I, including hydroxylation and deoxygenation; phase II reaction of picroside I, including glucuronidation, sulfation, and methylation; phase I biotransformations of metabolites, such as reduction and hydroxylation; and phase II biotransformations of metabolites, such as glucuronidation and sulfation. These results could offer insights into the effectiveness and toxicity of picroside I.

Simultaneous determination of gentiopicroside and its two active metabolites in rat plasma by LC-MS/MS and its application in pharmacokinetic studies.

Gentiopicroside is a natural secoiridoid glycoside that may require metabolic activation to exert pharmacological effects. In this study, two active metabolites of gentiopicroside (M1 and M2) were isolated from rat urines and identified with our previous method. Most importantly, a fast, sensitive and selective ultra high-performance liquid chromatography-tandem mass spectrometry method was developed to simultaneously determine gentiopicroside and its two metabolites in rat plasma. The analytes and internal standard (swertiamarin) were separated on an ACQUITY UPLC® BEH C18 column (2.1×50mm, 1.7µm) using gradient elution by acetonitrile and 0.1% formic acid at a flow rate of 0.4mL/min. The mass spectrometry detector was operated in the multiple reaction monitoring with positive ionization mode. The method had a good linearity over the concentration range of 0.2-10,000ng/mL for gentiopicroside and 0.1-5000ng/mL for the two metabolites. The validated method was successfully applied to the pharmacokinetic study of gentiopicroside and its metabolites after single oral administration of gentiopicroside (150mg/kg) to rats (n=8). The pharmacokinetic differences between gentiopicroside and its two metabolites were identified.Results provided the evidence for in vivo metabolism-based activation of gentiopicroside.

A systematic review of the protective role of swertiamarin in cardiac and metabolic diseases.

BACKGROUND: Swertiamarin, is a secoiridoid glycoside found in genera of Enicostemma Species (Enicostemma littorale and Enicostemma axillare) belonging to the family of gentianaceae, which has been reported to cure many diseases such as diabetes, hypertension, atherosclerosis, arthritis, malaria and abdominal ulcers. However, to the best of our knowledge, till date systematic studies to understand the molecular basis of cardiac and metabolic disease preventing properties of swertiamarin has not been reported. AIM OF THE REVIEW: The present review aims to compile an up-to-date information on the progress made in the protective role of swertiamarin in cardiac and metabolic diseases with the objective of providing a guide for future research on this bioactive molecule. MATERIALS AND METHODS: Information on the swertiamarin was collected from major scientific databases (Pubmed, Springer, google scholar, and Web of Science) for publication between1974-2016. In this review, the protective role of swertiamarin on cardiac and metabolic diseases was discussed. RESULTS: Swertiamarin reported to exhibit a wide range of biological activities such as anti-atherosclerotic, antidiabetic, anti-inflammatory and antioxidant effects. These activities were mainly due to its effect on various signaling pathways associated with cardiac remodeling events such as inhibition of NF-kB expression, LDL oxidation, apoptosis, inflammatory and lipid peroxidation markers and stimulation of antioxidant enzymes. CONCLUSION: Sweriamarin exhibit a wide range of biological activities. This review presents evidence supporting the point of view that swertiamarin should be considered a potential therapeutic agent against cardiac and metabolic diseases, giving rise to novel applications in their prevention and treatment.

Characterization of picroside II metabolites in rats by ultra-high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Picroside II, a bioactive compound isolated from Picrorhiza scrophulariiflora Pennell, has been reported to have hepatoprotective, neuroprotective, and antioxidant effects. However, the detailed in vivo biotransformation of this compound has been rarely reported. This study aimed to investigate the metabolic profiles of picroside II in rats by using ultra-high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Metabolite structures were elucidated based on accurate mass measurements of deprotonated molecules and their fragmentation patterns. Thirteen metabolites were structurally identified, and the detailed metabolic pathways were proposed. The findings revealed that after oral administration, picroside II mainly undergoes four metabolic pathways. In the first pathway, picroside II is deglycosylated to generate aglycone, which is isomerized to a dialdehyde-type intermediate. A series of metabolic reactions, including glucuronidation, subsequently occurs. In the second pathway, picroside II is subjected to ester bond hydrolysis to form vanillic acid, which is further subjected to sulfate conjugation, glycine conjugation, glucuronidation, and demethylation. In the third pathway, picroside II is directly conjugated with glucuronic acid to yield a predominant metabolite (M01) in plasma. In the fourth pathway, picroside II is directly conjugated with sulfate. These findings provide insights into the in vivo disposition of picroside II and are useful to understand the mechanism of effectiveness and toxicity of this compound as well as P. scrophulariiflora-related preparations.

Pharmacokinetics and tissue distribution of five active ingredients of Eucommiae cortex in normal and ovariectomized mice by UHPLC-MS/MS.

1. Pinoresinol di-O-ß-d-glucopyranoside (PDG), geniposide (GE), geniposidic acid (GA), aucubin (AN) and chlorogenic acid (CA) are the representative active ingredients in Eucommiae cortex (EC), which may be estrogenic. 2. The ultra high-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of the five ingredients showed good linearity, low limits of quantification and high extraction recoveries, as well as acceptable precision, accuracy and stability in mice plasma and tissue samples (liver, spleen, kidney and uterus). It was successfully applied to the comparative study on pharmacokinetics and tissue distribution of PDG, GE, GA, AN and CA between normal and ovariectomized (OVX) mice. 3. The results indicated that except CA, the plasma and tissue concentrations of PDG, GE, GA in OVX mice were all greater than those in normal mice. AN could only be detected in the plasma and liver homogenate of normal mice, which was poorly absorbed in OVX mice and low in other measured tissues. PDG, GE and GA seem to be better absorbed in OVX mice than in normal mice proved by the remarkable increased value of AUC0-∞ and Cmax. It is beneficial that PDG, GE, GA have better plasma absorption and tissue distribution in pathological state.

In vitro - In vivo metabolism and pharmacokinetics of picroside I and II using LC-ESI-MS method.

Picroside I and II, iridoid glycosides, are the major active markers of roots and rhizomes of Picrorhiza kurroa (family: Scrophulariaceae). The rhizomes of P. kurroa have been traditionally used to treat worms, constipation, low fever, scorpion sting, asthma and ailments affecting the liver. Various Ayurvedic and herbal preparations are available in the market which contains P. kurroa e.g. Arogyavadhini vati, Tiktadi kwath, Picrolax capsules and suspension. These preparations are used without any significant pharmacokinetics data. Previously, we have reported that oral bioavailability of picroside I and II is low. Most of the iridoid glycosides are primarily metabolized by intestinal microbial flora. So, it is necessary to determine the metabolic profile of picroside I and II and check the correlation with lower bioavailability. Therefore, this study was designed to check metabolic (in vitro and in vivo) profile along with pharmacokinetic profile of picroside I and II. For this, a sensitive and selective LC-ESI-MS method was developed and validated for simultaneous determination of picroside I and II in rat plasma. Chromatographic separations were performed on C18 column. The mobile phase consisted of acetonitrile: 10 mM ammonium acetate buffer [90:10 v/v], pH 3.5. In-vitro Metabolic study was performed on rat liver microsomes and primary hepatocytes. In-vivo pharmacokinetic and metabolic profile of picroside I and II was generated after oral administration of Kutkin (mixture of picroside I and II) to Sprague-Dawley rats. Various pharmacokinetic parameters viz. Cmax, Tmax, AUC(0-t) were determined. In metabolic study, eight metabolites of picroside I and six metabolites of picroside II were identified in vitro, out of which four metabolites for each picroside I and picroside II were identified in vivo.

[Effect of D-cellobiose on oral bioavailability of gentiopicroside].

In this study, the effect of D-cellobiose on oral bioavailability of gentiopicroside (GPS) was investigate. The influence of D-cellobiose on GPS was achieved by calculating the residual GPS after being degraded with ß-glucosidase or intestinal flora, and the data demonstrated D-cellobiose could inhibit the degradation of GPS in intestines; in bioavailability experiment, D-cellobiose could significantly improve the oral bioavailability (P<0.05) of GPS at the mass ratio of 1∶5, 1∶10 (GPS-D-cellobiose). D-cellobiose applied in this study may improve the oral bioavailability of GPS through delaying the degradation in intestines.

[Borneol promotes oral absorption and penetration into brain of puerarin and catalpol].

To investigate the effect of borneol on the oral absorption and penetration into brain of puerarin and catalpol from cell level and animal level, and screen the concentration of borneol that is suitable for Zige compound oral preparation. Blood-brain barrier(BBB) model was established by co-culture of primary brain microvessel endothelial cells(BMEC) and astrocytes(As) in rats, and it was used to investigate the effect of borneol(concentration from 6.25 to 100 mg•L⁻¹) on the transport of puerarin and catalpol. The pharmacokinetics of puerarin and catalpol in plasma and brain of rats were compared after intragastric administration of borneol solution (0, 25, 50 and 100 mg•kg⁻¹) immediately followed by puerarin(200 mg•kg⁻¹) and catalpol(45 mg•kg⁻¹) nanocrystal suspension. Barrier function was basically formed after co-culturing of brain microvascular endothelial cells and astrocytes for 7 d. The permeability of puerarin and catalpol across blood-brain barrier was increased significantly(P<0.05) and transendothelial electrical resistance(TEER) values at 2 h were decreased significantly(P<0.01) when the concentration of borneol was between 12.5 to 100 mg•L⁻¹ as compared with the control group. Borneol at the dose of 50 mg•kg⁻¹ and 100 mg•kg⁻¹ could significantly increase the oral absorption of puerarin(P<0.05), but there was no obvious effect for catalpol. AUCbrain/AUCblood for puerarin was highest with borneol at dose of 100 mg•kg⁻¹ (P<0.05), while AUCbrain/AUCblood for catalpol was highest with borneol at dose of 50 mg•kg⁻¹ (P<0.05). AUCbrain was highest at 100 mg•kg⁻¹ for puerarin(P<0.05); while for catapol, it was highest at 50 mg•kg⁻¹, but it was not significantly different from 100 mg•kg⁻¹. In conclusion, borneol could increase the amount of puerarin and catalpol in brain after oral administration and the optimized dose shall be 100 mg•kg⁻¹.

[Effects of different preparation technologies on concentrations of puerarin and catalpol in plasma and brain of rats after oral administration].

To compare the effects of different preparation technologies on the concentrations of puerarin and catalpol in plasma and brain of rats after oral administration, in order to lay an experimental basis for developing new oral Zige preparations. The nanocrystal, self-microemulsions (tween-80 and Cremophor RH-40 as emulsifiers) and inclusion complex of HP-ß-CD containing puerarin and catalpol were prepared. The concentrations of puerarin and catalpol in plasma and brain of rats after oral administration were determined by HPLC-MS/MS method. The pharmacokinetic parameters and brain target index were compared. The results showed that preparation technologies had different influences on the concentrations of puerarin and catalpol in plasma and brain. The self-microemulsion (tween-80) could significantly increase the oral absorption of puerarin than other technologies(P<0.05), and inclusion complex could remarkably increase the oral absorption of catalpol than nanocrystal(P<0.01). For puerarin, the brain targeting index of inclusion complex was the highest (P<0.05); but for catalpol, the brain targeting index of inclusion complex and self-microemulsions were both higher than nanocrystal (P<0.05). The self-microemulsion(tween-80) had the highest AUCbrain of puerarin than other groups (P<0.01); the inclusion complex had the highest AUCbrain for catalpol, but there was no significant difference compared with self-microemulsions. In conclusion, the self-microemulsion (tween-80) technology could increase the amount of puerarin and catalpol in brain, and was expected to be used in new oral Zige preparations.

Pharmacokinetics and tissue distribution of Aucubin, Ajugol and Catalpol in rats using a validated simultaneous LC-ESI-MS/MS assay.

To enable an investigation of pharmacokinetics and tissue distribution of Aucubin, Ajugol and Catalpol in rats, a high-performance liquid chromatography-electro spray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed for the simultaneous quantitative determination of the three compounds. Biological samples were prepared by a simple protein precipitation with methanol (containing 0.05% formic acid). The analytes were separated by a C18 reversed phase column and detected with a triple quadrupole tandem mass spectrometer in the multiple-reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 364.3→149.0 for Aucubin, m/z 366.5→151.1 for Ajugol, m/z 380.0→183.3 for Catalpol and m/z 530.3→183.1 for Picroside-II (IS) in positive ionization. Good linearity of each calibration curve was produced over the concentration range of 1-1000ng/mL. The lower limit of quantification (LLOQ) was 1ng/mL for the three analytes. This method was successfully applied to the pharmacokinetic and tissue distribution studies of Aucubin, Ajugol and Catalpol in rat. The current results revealed pharmacokinetic behaviors of the herb compound and provided novel evidence of the presence of Aucubin and Catalpol in rat brain. The acquired data would be helpful for the clinical application and further studies of Traditional Chinese Medicines (TCM) with active ingredients of Iridoid Glycosides.

Comparative pharmacokinetics of catalpol and acteoside in normal and chronic kidney disease rats after oral administration of Rehmannia glutinosa extract.

In this study, a sensitive and robust ultra-performance liquid chromatography-mass spectrometry method with multiple-reaction monitoring mode was developed, validated, and applied to determine pharmacokinetics of catalpol and acteoside in normal and doxorubicin-induced chronic kidney disease rats after oral administration of Rehmannia glutinosa extract. The lower limits of quantification for catalpol and acteoside in rat plasma were 2.62 and 0.61 ng/mL, with a signal-to-noise ratio of ≥10. Precision and accuracy studies showed that catalpol and acteoside plasma concentrations were within the 10% range in all studies. The extraction recoveries of catalpol and acteoside were both >68.24% and the matrix effects ranged from 96.59 to 101.62%. The method was successfully applied to the pharmacokinetic study of catalpol and acteoside after oral administration of RG extract to normal and model rats, respectively. This study might further support the traditional use of RG to treat kidney diseases clinically.

A pre-clinical pharmacokinetic study in rats of three naturally occurring iridoid glycosides, Picroside-I, II and III, using a validated simultaneous HPLC-MS/MS assay.

A selective and sensitive high-performance liquid chromatography-electro-spray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the simultaneous quantitative determination of Picroside-I, II, and III in rat plasma and tissue homogenate to aid the pre-clinical studies. The chromatographic separation was performed on a Hypersil GOLD AQ C18 column using a gradient elution program with a mobile phase consisting of 2mM ammonium acetate and acetonitrile. The detection was achieved using a triple quadrupole tandem MS in negative ionization multiple reaction monitoring (MRM) mode. One-step protein precipitation was selected for plasma and tissue sample preparation while liquid-liquid extraction failed to achieve satisfactory recoveries. The calibration curves of all three analytes in either plasma or tissue homogenate showed good linearity over the concentration range of 0.5-500ng/mL with a limit of quantitation at 0.5ng/mL. Both the intra- and inter-day accuracy and precision were within ±10%. The extraction recoveries were >70%, and the relative matrix effect ranged from 80.4% to 107.4% in all the biological samples. All the analytes were stable in matrices for at least 24h at room temperature, or 21 days in frozen. Three freeze/thaw cycles did not cause degradation. The method was successfully applied for quantification of the three iridoid glycosides in the collected plasma and various tissues following intravenous administration in rats. Picroside-I, II, and III were all eliminated rapidly with large volume of distribution. Among the three glycosides, Picroside-II showed the highest liver uptake, and only Picroside-I and II were found to get across the blood brain barrier (BBB). These results were consistent with their hepatoprotective or neuroprotective effects reported clinically. With the aid of the efficient and reliable simultaneous LC-ESI-MS/MS assay this pharmacokinetic study provided insights into their therapeutic targets of these three iridoid glycosides as well as valuable experimental basis for an expansion of their clinical indications.

Determination and pharmacokinetic study of gentiopicroside, geniposide, baicalin, and swertiamarin in Chinese herbal formulae after oral administration in rats by LC-MS/MS.

A sensitive and efficient liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of gentiopicroside, geniposide, baicalin, and swertiamarin in rat plasma. To avoid the stress caused by restraint or anesthesia, a freely moving rat model was used to investigate the pharmacokinetics of herbal medicine after the administration of a traditional Chinese herbal prescription of Long-Dan-Xie-Gan-Tang (10 g/kg, p.o.). Analytes were separated by a C18 column with a gradient system of methanol-water containing 1 mM ammonium acetate with 0.1% formic acid. The linear ranges were 10-500 ng/mL for gentiopicroside, geniposide, and baicalin, and 5-250 ng/mL for swertiamarin in biological samples. The intra- and inter-day precision (relative standard deviation) ranged from 0.9% to 11.4% and 0.3% to 14.4%, respectively. The accuracy (relative error) was from -6.3% to 10.1% at all quality control levels. The analytical system provided adequate matrix effect and recovery with good precision and accuracy. The pharmacokinetic data demonstrated that the area under concentration-time curve (AUC) values of gentiopicroside, geniposide, baicalin, and swertiamarin were 1417 ± 83.8, 302 ± 25.8, 753 ± 86.2, and 2.5 ± 0.1 min µg/mL. The pharmacokinetic profiles provide constructive information for the dosage regimen of herbal medicine and also contribute to elucidate the absorption mechanism in herbal applications and pharmacological experiments.

Application of a liquid chromatography-tandem mass spectrometry method to the pharmacokinetics, tissue distribution and excretion studies of sweroside in rats.

A sensitive, reliable and accurate high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) was developed and validated for the quantification of sweroside in rat plasma, tissue and excretion. A single-step protein precipitation by methanol was used to prepare samples. Sweroside and swertiamarin (internal standard, IS) were separated by using a C18 column and a mobile phase consisted of methanol and water containing 0.1% formic acid running at a flow rate of 0.8ml/min for 6min. Detection and quantification were performed using a mass spectrometer by the multiple-reaction monitoring (MRM) in positive electrospray ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were [M+H](+)359.1→197.2 for sweroside and [M+Na](+)397.4→165.3 for swertiamarin (IS), respectively. The inter-day precision (RSD %) was less than 11.20% and intra-day precision (RSD %) was less than 10.90%, while the inter-day accuracy (RE %) was ranged from -9.69 to 9.17% and intra-day accuracy (RE %) was ranged from -10.56 to 13.47%. The mean elimination half-life (t1/2) of sweroside for 5, 10 and 15mg/kg dose were 78.8, 67.6 and 77.2min, respectively. And sweroside follows linear plasma pharmacokinetics across the investigated dosage range in rats (5-15mg/kg). The absolute bioavailability (F %) of sweroside was 11.90% on average. The results of tissue distribution showed the higher sweroside concentrations were found in kidney, liver, spleen and lung, and the small amount of drug was distributed into the brain tissue. The high distribution in liver confirms the reports that sweroside has hepatoprotective activity and promoted liver regeneration, and there was no long-term accumulation of sweroside in rat tissues. Total recoveries of sweroside within 48h were 0.67% in bile, 1.55% in urine and 0.46% in feces, which might be resulted from liver first-pass effect. The above results suggested that sweroside was mainly excreted as the metabolites.