PK-PD Correlation of Erigeron Breviscapus Injection in the Treatment of Cerebral Ischemia-Reperfusion Injury Model Rats.

By measuring the cerebral infarction rate and neurological behavioral score of rats in a sham operation group, an MCAO model control group and an Erigeron breviscapus injection treatment group, we explored the therapeutic effects of Erigeron breviscapus injection on brain tissue and neuroethological injury in rats. Plasma samples were collected at 18 time points after intravenous injection of Erigeron breviscapus. The levels of scutellarin, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, chlorogenic acid and isochlorogenic acid B in rat plasma at the various time points were determined by an HPLC method, and drug concentration versus time plots were constructed to estimate the pharmacokinetic parameters. Finally, a PK-PD combined model was used to analyze the relationship between the blood concentration, time and therapeutic effects of the seven active components. The results of the pharmacodynamics studies showed that the cerebral infarction rate of rats in the Erigeron breviscapus injection group decreased significantly at 5 min, 10 min, 20 min, 6 h, 8 h, 18 h, 24 h, 32 h, 40 h and 48 h after cerebral ischemia. Abnormal neurological behavior scores were significantly reduced after 4 h of cerebral ischemia. The pharmacokinetics results showed that the seven chemical constituents in Erigeron breviscapus injection reached their highest detection value after 5 min of cerebral ischemia. The lowest detection values of scutellarin and isochlorogenic acid B appeared after 6 h of cerebral ischemia but could not be detected after 8 h. The lowest detection values of 5-caffeoylquinic acid and 4,5-dicaffeoylquinic acid were found in the third hour of cerebral ischemia but not after 4 h. The lowest detection values of 4-caffeoylquinic acid, 3,5-dicaffeoylquinic acid and chlorogenic acid were found during the second hour of cerebral ischemia but not at the third hour. However, at 18 h, 24 h, 32 h and 40 h of cerebral ischemia, the cerebral infarction rates of rats in the Erigeron breviscapus injection group were significantly reduced, with decreased values of 6.22%, 11.71%, 6.92% and 4.96%, respectively, and the effects were stronger than those after 5-20 min of cerebral ischemia. The decreased values reached their highest value after 24 h of cerebral ischemia. Our results show that the effects of Erigeron breviscapus injection on reducing the cerebral infarct rate in MCAO model rats are characterized by a fast onset and long maintenance time. The 5-min blood concentration in cerebral ischemia was the highest test value, and after this time, the cerebral infarction rate of MCAO rats began to decrease. However, the peak value of the effects lagged behind that of the plasma concentration. The maximum effective time for Erigeron breviscapus injection appeared 24 h after cerebral ischemia, which provides a reference for the screening of specific drugs for ischemic stroke, optimal dosing regimens and rational clinical drug use. Graphical Abstract.

ß-Glucuronidase- and OATP2B1-mediated drug interaction of scutellarin in Dengzhan Xixin Injection: A formulation aspect.

Scutellarin is the major and active constituent of Dengzhan Xixin Injection (DZXX), a traditional Chinese medicine prepared from the aqueous extract of Erigeron breviscapus and widely used for the treatment of various cerebrovascular diseases in clinic. In present study, the possible pharmacokinetic differences of scutellarin after intravenous administration of scutellarin alone or DZXX were explored. Additional, the potential roles of ß-glucuronidase (GLU) and OATP2B1 in drug-drug interaction (DDI) between scutellarin and constituents of DZXX were further evaluated in vitro. The plasma concentration, urinary and biliary excretion of scutellarin in rats after administration of DZXX, were significantly higher than those received scutellarin, while pharmacokinetic profile of Apigenin 7-O-glucuronide (AG) in rats was similar no matter AG or DZXX group. Furthermore, higher concentration in brain and plasma, however, lower level of scutellarin in intestine were observed after intravenous administration of DZXX. Finally, AG and caffeoylquinic acid esters were found to significantly inhibit GLU and OATP2B1 in vitro, which might explain, at least in part, the pharmacokinetic DDI between scutellarin and other chemical constituents in DZXX. The findings provided deep insight into the prescription-formulating principle in DZXX for treating the cerebrovascular diseases.

Identification and characterization of chemical constituents in Dengzhan Shengmai Capsule and their metabolites in rat plasma by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Dengzhan Shengmai Capsule (DZSMC) is a traditional Chinese medicine (TCM) formula with remarkable clinical effect in the treatment of stroke sequelae. Exploring the components of DZSMC and detecting the absorbed prototype constituents and metabolites in blood are of great significance to clarify the effective substances of this prescription. Here, a reliable method using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was established for the comprehensive analysis of chemical constituents of DZSMC and their metabolites in rat plasma after gastric perfusion. Two acquisition modes, including MSE mode and Fast DDA mode, were performed for acquiring more precursor ions and cleaner precursor-product ions background during the study of constituents of DZSMC. As a result, a total of 125 constituents were unambiguously characterized or tentatively identified. For the first time, a total of 92 components, including 44 prototype components and 48 metabolites were unambiguously or tentatively identified in rat plasma. The metabolic pathways included phase I reactions (hydration, hydrogenation, oxidation, demethylation and hydroxylation) and phase II reactions (conjugation with glucuronide, sulfate and methyl). Furthermore, the metabolites from caffeic acid and scutellarin were characterized and validated by phase II metabolic reactions in vitro, which could be established as a simulated in vivo environment of metabolites identification and verification of TCM formula. It is the first systematic study on metabolism of DZSMC in vivo and could also provide a valid analytical strategy for characterization of the chemical compounds and metabolites of TCM formula.

Ultra-high-performance liquid chromatography with tandem mass spectrometry method for determination of four compounds in rat plasma after oral administration of Xanthii fructus and stir-fried Xanthii fructus extracts.

Xanthii fructus (XF), the fruit of Xanthium sibiricum Patr., is a traditional Chinese materia medica commonly used to treat allergic rhinitis and other rhinitis diseases. To uncover the mechanism of the stir-frying process and its effect on the pharmacokinetic behavior of active compounds in model rats, four active compounds-chlorogenic acid, 4-caffeoylquinic acid, 1,5-O-dicaffeoylquinic acid and apigenin-were selected based on previous spectrum-effect experiments. High performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-QqQ-MS) technology, an accurate and feasible method, was applied to measure the concentration of these four compounds in rat plasma. This validated method can accurately measure the concentration of each compound at each sampling point of rat plasma. This validated method shows good linearity, extraction recoveries, matrix effects, intra- and inter-day precision and stabilities. Compared with the XF group, the maximum plasma concentration (Cmax ) value of 1,5-O-dicaffeoylquinic acid decreased remarkably (p < 0.05) after oral administration of stir-fried Xanthii fructus (SXF) extract, while the other compounds showed no significant difference. The mean residence time value of chlorogenic acid (p < 0.05) and 1,5-O-dicaffeoylquinic acid (p<0.01) after oral administration of SXF extraction demonstrated significant differences compared with the XF group, while the other two compounds showed no statistical difference, indicating that the stir-frying process prolonged the effect time and delayed the removal time of chlorogenic acid and 1,5-O-dicaffeoylquinic acid. The values of the area under the plasma concentration-time curve from zero to the last quantifiable time-point, the area under the plasma concentration-time curve from zero to infinity, the time to maximum concentration and the elimination half-life of four compounds in the SXF group showed no statistically significant difference from the XF group. From this data, we speculated that the stir-frying process can not only keep the absorption of 4-caffeoylquinic acid and apigenin, but also increase the effect time of chlorogenic acid and 1,5-O-dicaffeoylquinic acid, which could be the mechanism underlying the stir-frying process enhancing the effects of XF.

LC-MS/MS method for the simultaneous quantification of luteolin, wedelolactone and apigenin in mice plasma using hansen solubility parameters for liquid-liquid extraction: Application to pharmacokinetics of Eclipta alba chloroform fraction.

Eclipta alba (Bhringraj) in ayurveda has been widely used as a traditional medicine for its multi-therapeutic properties for ages. Luteolin (LTL), wedelolactone (WDL) and apigenin (APG) are the three main bioactive phytochemicals present in Eclipta alba extract. However there was a lack of sensitive bioanalytical method for the pharmacokinetics of these free compounds in plasma which majorly contributes for their activities after oral administration of Eclipta alba. The present study aims to develop a sensitive, rapid and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous estimation of mice plasma concentrations of LTL, WDL and APG using quercetin as an internal standard for the pharmacokinetic analysis. Analytes were separated on Phenomenex Luna C18 (150 × 4.6 mm, 3.0 µm) column with mobile phase containing methanol: acetonitrile (90: 10, v/v) and 0.1% formic acid in 10 mM ammonium formate buffer in the ratio of 70: 30 (v/v) in isocratic mode. Liquid-liquid extraction was optimized using Hansen solubility parameters and diethyl ether finalized as an extraction solvent for the recovery ranging from 61 to 76% for all analytes in mice plasma. The validated method has an accuracy and precision over the linearity range of 0.1-200 ng/mL with a correlation coefficient (r2) of ≥0.997. The intra and inter-day assay accuracy was between 98.17 and 107% and 95.83-107.89% respectively and the intra and inter day assay precision ranged from 0.37-6.05% and 1.85-10.76%, respectively for all the analytes. This validated method can be used for future clinical investigation studies of Eclipta alba extracts.

Antithrombotic and antiplatelet activities of vicenin-2.

Cyclopia subternata is a medicinal plant commonly used in traditional medicine to relieve pain in physiological processes. However, there are limited modern data on its pharmacological effects and active components relating to its traditional use. Here, the anticoagulant and antiplatelet activities of vicenin-2 (VCN), an active compound in C. subternata, were determined. The anticoagulant activities were investigated by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of thrombin and activated factor X (FXa). The effects of VCN on the expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated in tumor necrosis factor (TNF)-α-activated human umbilical vein endothelial cells (HUVECs). Treatment with VCN resulted in prolonged aPTT and PT and inhibition of the activities of thrombin and FXa, as well as inhibited production of thrombin and FXa in HUVECs. In addition, VCN inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. VCN also elicited anticoagulant effects in mice. In addition, treatment with VCN resulted in significant reduction of the PAI-1 to t-PA ratio. Collectively, VCN possesses antithrombotic activities and offers a basis for development of a novel anticoagulant.

Clinical tolerability and pharmacokinetics of Erigerontis hydroxybenzene injection: results of a randomized phase I study in healthy Chinese volunteers.

Multiple phenolic compounds in the extract of Erigeron breviscapus synergistically contribute to the neurovascular protective effects. We conducted a phase I and pharmacokinetic study with the phenolic compound-enriched product extracted from Erigeron breviscapus, Erigerontis hydroxybenzenes injection (EHI), in healthy Chinese volunteers. A randomized, open-label, single-center, double-arm, dose-escalation study of EHI was conducted. The tolerability of intravenously EHI administrated in single- or multiple-dose (once daily for 7 days) was studied in 40 healthy Chinese volunteers and the pharmacokinetics of EHI was studied in additional 10 volunteers. The tolerated dose of intravenous infusion of EHI in healthy Chinese volunteers was 6 vials (equivalent to 90 mg bioactive phenolic compounds). The main limitations to dose escalation of EHI were transit changes in electrocardiogram and mild, transit increase in alanine aminotransferase. After intravenous administration of EHI, the average systemic clearance of multiple phenolic compounds of scutellarin, 1,3-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 3,4-dicaffeoylquinic acid were 131, 29, 262, 112 L/h for male volunteers and 202, 28, 252, 117 L/h for female volunteers. The intervention of intravenous infusion of EHI in healthy Chinese volunteers was generally tolerated. The findings from this study provide data on the tolerability and pharmacokinetics of the extract from Erigeron breviscapus and support further trials.

Integrated pharmacokinetics and biodistribution of multiple flavonoid C-glycosides components in rat after oral administration of Abrus mollis extract and correlations with bio-effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Abrus mollis, a commonly used traditional Chinese medicine in China and other Asia countries, has been used clinically to prevent and treat hepatitis and alcoholic liver disease for decades. MATERIALS AND METHODS: A modified HPLC-MS method was developed for the determination of vicenin-2 (AM-I), isoschaftoside (AM-II), and schaftoside (AM-III) of AM extract (AME) in rat plasma and tissues (heart, liver, spleen, lungs, and kidneys). Following oral administration of AME to rat at a dose of 200mg/kg, the concentrations of AM-I, II and III in plasma and tissues were quantified. An integrated double peak pharmacokinetics model was used to fit the concentration-time curves. The effects of drug on the bile flow and toe swelling of rats induced by carrageenan were also studied. RESULTS: The limit of quantitation of this modified HPLC-MS method decreased from 25 to 5ng/mL for plasma and from 100 to 10ng/g for tissue. These concentration-time curves show two successive maximum concentrations. The results of integrated double peak pharmacokinetics in this paper indicated that the three flavonoid C-glycosides may be absorbed by two sites of intestine in vivo. These results of bile flow and toe swelling showed a significant correlation between the pharmacokinetics and pharmacodynamics. CONCLUSIONS: The novel integrated double peak pharmacokinetic approach to studying the holistic pharmacokinetic properties of traditional Chinese medicine has been successfully developed and validated using AM as a model drug. This study would be a useful guide for the holistic double peak pharmacokinetic study in consistence with the intrinsic theory and characteristics of traditional Chinese medicine.

Simultaneous determination of scutellarin and tetrahydropalmatine of Deng-yan granule in rat plasma by UFLC-MS/MS and its application to a pharmacokinetic study.

Deng-yan granule, consisting of Herba Erigerontis Breviscapi, Rhizoma Corydalis Yanhusuo and Radix Astragali Mongolici, is a widely used Traditional Chinese Medicine preparation for treatment of coronary heart disease. Scutellarin and tetrahydropalmatine are main active constituents in Herba Erigerontis Breviscapi and Rhizoma Corydalis Yanhusuo, and have been used as marker components for quality control of Deng-yan preparations. In order to make good and rational use of Deng-yan granule in the future, a rapid, sensitive and high throughput ultra-fast liquid chromatography with tandem mass spectrometry (UFLC-MS/MS) method was developed for the simultaneous determination of scutellarin and tetrahydropalmatine in rat plasma using rutin as internal standard (IS). The plasma samples were extracted by liquid-liquid extraction with ethyl acetate after acidification and separated on a Shim-pack XR-ODS C18 column (75mm×3.0mm, 2.2µm) with a mobile phase consisting of methanol-0.1% formic acid water (50:50, v/v) at a flow rate of 0.4mL/min. Mass spectrometric detection was conducted on an API 3200 QTRAP mass spectrometry equipped with electrospray ionization source in positive ionization mode. Quantification was performed using multiple reaction monitoring (MRM) by monitoring the fragmentation of m/z 463.2→287.1 for scutellarin, m/z 356.1→192.1 for tetrahydropalmatine and m/z 611.2→303.2 for IS, respectively. The linear range was 10-5000ng/mL for both scutellarin and tetrahydropalmatine with lower limit of quantitation (LLOQ) of 10ng/mL. The intra- and inter-day precisions were below 12.2% for scutellarin and below 9.7% for tetrahydropalmatine in terms of relative standard deviation (RSD), and the accuracy was within ±9.1% for scutellarin and within ±11.2% for tetrahydropalmatine in terms of relative error (RE). Extraction recovery, matrix effect and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of scutellarin and tetrahydropalmatine after oral administration of Deng-yan granule to rats.

Pharmacokinetic interaction between scutellarin and valsartan in rats.

Scutellarin is the main effective constituent of breviscapine, a flavonoid mixture isolated from the dried whole plant of Erigeron breviscapus (Vant.) Hand-Mazz, and valsartan is used as an antihypertensive drug. These two drugs have already been clinically used together to treat diabetic nephropathy (DN) in China, and the combined medications showed some enhanced protection against DN. The aim of this study is to investigate the potential pharmacokinetic interaction between scutellarin and valsartan in rats. Breviscapine injection (20 mg x kg(-1), i.v.) and valsartan (15 mg x kg-, i.g.), either alone or together were given to 18 male Sprague-Dawley rats. Concentrations of scutellarin and valsartan were quantified by HPLC, and pharmacokinetic parameters were calculated by non-compartmental methods. We found that the pharmacokinetic parameters of scutellarin altered significantly after co-administration of oral valsartan. The plasma clearance (CL(p)) and the bile clearance (CL(b)) of scutellarin were reduced significantly in the presence of valsartan. After oral administration of valsartan with or without intravenous scutellarin, however, the pharmacokinetic parameters of valsartan were comparable. In conclusion, our data suggests that the concurrent use of valsartan reduces the biliary excretion of scutellarin, and this may be due to the inhibitory effect of valsartan on the biliary excretion of scutellarin mediated by Mrp2 (Multidrug resistance-associated protein 2).

Caecal absorption of vitexin-2-O-xyloside and its aglycone apigenin, in the rat.

The in vivo bioavailability of the flavone-C-glycosides has been little studied compared to their O-glycoside analogues, which are both more common in nature and considered more easily hydrolyzed than C-glycosides, by both enterocytes and gut microbiota. In this study, we used vitexin-2-O-xyloside (VOX), an apigenin-8-C-glucoside-2-O-xyloside, purified from seeds of Swiss chard (Beta vulgaris cicla), to investigate VOX absorption into portal blood compared to its aglycone, apigenin. We used a rat model in which we ligated the ileo- and colo-caecal junctions, then administered apigenin or VOX directly into the caecum. Blood samples were drawn from the portal vein at timed intervals over 40 min. The kinetic profile of appearance in portal blood of the compounds and their metabolites was evaluated by HPLC-ESI-MS. Apigenin was found in portal blood both as the aglycone and as an apigenin-glucuronide derivative. The VOX was found unchanged and as a reduced monoglycoside, which underwent glucuronidation. By collecting the bile, we confirmed that the liver received unchanged VOX, which was returned to the gut by enterohepatic recirculation for reabsorption from the ileum. The amount of apigenin and VOX remaining in the caecum accounted for ∼15% and ∼26%, respectively. These data show for the first time that the C-glycoside VOX is absorbed unchanged and undergoes enterohepatic recirculation in addition to hydrolysis to the monoglycoside, reduction and conjugation to form a bioavailable glucuronide.

Simultaneous determination of vitexin and isovitexin in rat plasma after oral administration of Santalum album L. leaves extract by liquid chromatography tandem mass spectrometry.

An LC-MS/MS method was developed for the simultaneous determination of vitexin and isovitexin in rat plasma, using puerarin as the internal standard (IS). Plasma samples extracted with protein precipitation procedure were separated on a Diamonsil® C(18) column (150 × 4.6 mm, 5 µm) with a mobile phase composed of methanol and 0.1% formic acid (45:55, v/v). The detection was accomplished by multiple reaction monitoring mode in positive electrospray ionization source. The optimized mass transition ion-pairs for quantitation were m/z 431.2 → 311.1 for vitexin and isovitexin, and m/z 415.1 → 295.1 for IS. The total run time was 7.5 min for each injection. The calibration curves were linear (r(2) > 0.99) over the investigated concentration range (2.00-2000 ng/mL) and the lower limits of quantification were 2.00 ng/mL in rat plasma sample. The intra- and inter-day relative standard deviations were no more than 14.9% and the relative errors were within the range of -3.2-2.1%. The extraction recoveries for both compounds were between 89.3 and 97.3%. The robust LC-MS/MS method was further applied in the pharmacokinetic study in Sprague-Dawley rats after oral administration of Santalum album L. leaves extract at a dose of 116 mg/kg.

[Determination of vitexin in plasma by HPLC-MS/MS method and its pharmacokinetics in rats].

OBJECTIVE: To establish a HPLC-MS/MS method for the determination of vitexin in rat plasma and its pharmacokinetics. METHODS: The HPLC-MS/MS method used Capcell Pak C18 column (50 mm x 2.0 mm I. D., 5 microm). The mobile phase was methanol and water (95:5, V/V, containing 0.1% formic acid) at a flow rate of 0.2 mL/min. Electrospray ionization (ESI) in negative ion mode and multiple reaction monitoring (MRM) was used for the quantification of vitexin with a monitored transitions m/z 431-->311 for vitexin and m/z 269-->225 for internal standard (I. S., emodin). RESULTS: Linear calibration curves were obtained over the concentration range of 0.5-2000 ng/mL (r = 0.9960) with the lowest limit of quantification (LLOQ) of 0.5 ng/mL. The recovery was in the range of 76.1%-89.0%. The relative standard deviations for the intra-day and inter-day validation were less than 11%. CONCLUSION: The method is simple, accurate, fast, sensitive and suitable for the pharmacokinetic study of vitexin in rats.

Influences of glucose on the dietary hydroxyflavonoid-plasma protein interaction.

The influence of glucose on the interaction between flavonoids and plasma proteins from healthy humans (HPPs) was investigated. Glucose affected the flavonoid-protein interactions depending upon their structures. Glucose significantly reduced the affinities of HPPs for 6-hydroxyflavone by 10.72 times, slightly weakened the affinities of HPPs for quercetin, 7-hydroxyflavone, and kaempferol, and hardly affected the affinities of HPPs for myricetin, chrysin, and 3,7-dihydroxyflavone on the first day. However, glucose obviously enhanced the affinities of HPPs for 3-hydroxyflavone, luteolin, and apigenin. Glucose significantly weakened the binding affinities of HPPs for chrysin, kaempferol, quercetin, and myricetin by 6.17, 7.94, 14.12, and 112.2 times, when kept at 37 °C under air conditions for 14 days, and the binding affinities of HPPs for 7-hydroxyflavone, luteolin, 3,7-dihydroxyflavone, 3-hydroxyflavone, and 6-hydroxyflavone were slightly decreased by 1.35-, 1.58-, 1.58-, 1.9-, and 2.4-fold. The binding affinity between apigenin and HPP was hardly influenced. Glucose weakened the binding affinities of HPPs for hydroxyflavonoids. The differences between log K(a)(absence) and log K(a)(presence) were bigger for the more lipophilic hydroxyflavonoids, and more lipophilic hydroxyflavonoids are easily affected by glucose, when kept at 37 °C under air conditions for 14 days. These flavonoids with lower hydrogen donor/acceptor numbers prefer to stably interact with HPPs in the presence of glucose. However, other flavonoids with high hydrogen donor/acceptor numbers (multi-hydroxyl flavonoids) were apt to reduce their affinities with HPPs in the presence of glucose.

Mechanistic studies on the absorption and disposition of scutellarin in humans: selective OATP2B1-mediated hepatic uptake is a likely key determinant for its unique pharmacokinetic characteristics.

Scutellarin [scutellarein-7-O-glucuronide (S-7-G)] displayed a unique pharmacokinetic profile in humans after oral administration: the original compound was hardly detected, whereas its isomeric metabolite isoscutellarin [scutellarein-6-O-glucuronide (S-6-G)] had a markedly high exposure. Previous rat study revealed that S-7-G and S-6-G in the blood mainly originated from their aglycone in enterocytes, and that the S-7-G/S-6-G ratio declined dramatically because of a higher hepatic elimination of S-7-G. In the present study, metabolite profiling in human excreta demonstrated that the major metabolic pathway for S-6-G and S-7-G was through further glucuronidation. To further understand the cause for the exposure difference between S-7-G and S-6-G in humans, studies were conducted to uncover mechanisms underlying their formation and elimination. In vitro metabolism study suggested that S-7-G was formed more easily but metabolized more slowly in human intestinal and hepatic microsomes. Efflux transporter study showed that S-6-G and S-7-G were good substrates of breast cancer resistance protein and multidrug resistance-associated protein (MRP) 2 and possible substrates of MRP3; however, there was no preference great enough to alter the S-7-G/S-6-G ratio in the blood. Among the major hepatic anion uptake transporters, organic anion-transporting polypeptide (OATP) 2B1 played a predominant role in the hepatic uptake of S-6-G and S-7-G and showed greater preference for S-7-G with higher affinity than S-6-G (K(m) values were 1.77 and 43.9 µM, respectively). Considering the low intrinsic permeability of S-6-G and S-7-G and the role of OATP2B1 in the hepatic clearance of such compounds, the selective hepatic uptake of S-7-G mediated by OATP2B1 is likely a key determinant for the much lower systemic exposure of S-7-G than S-6-G in humans.

[Simultaneous determination of isoorientin, scutellarin and cynaroside in rat plasma and pharmacokinetics by UPLC-MS/MS].

OBJECTIVE: To establish a UPLC-MS/MS analysical method for simultaneous determination of concentrations of isoorientin, scutellarin and cynaroside in rat plasma and to study their pharmacokinetic characteristics after intravenous injection of 3 doses of Fufang Hongcao in rats. METHOD: Acidified plasma samples were precipitated for protein with methanol. Waters Acquity BEH C18 column was adopted for spectrum, with mobile phase as 0. 1% formic acid acetonitrile-0. 1% formic acid-water gradient elution. Detection was carried out by the multiple reaction monitoring (MRM) positive ion mode with ESI ionization source. RESULT: Three flavonoids show a good linear relationship, with the extraction recovery ranging between 78.56% and 101.91% and a high intra-and inter-day precisions and accuracy. The MRT of the three flavonoids were all lower than 22 min in rats. CONCLUSION: The above men tioned method is so specific, rapid, sensitive that it is suitable for pharmacokinetic studies of Fufang Hongcao injection in rats.

LC determination of luteolin-7-O-ß-D-glucoside and apigenin-7-O-ß-D-glucoside in rat plasma after administration of Humulus scandens extract and its application to pharmacokinetic studies.

The present study was to investigate the pharmacokinetics of luteolin-7-O-ß-D-glucoside (LGL) and apigenin-7-O-ß-D-glucoside (AGL) in rat plasma after intravenous administration of the Humulus scandens extract (HSE). A simple and accurate high-performance liquid chromatographic (HPLC) method was successfully developed for simultaneous determination of LGL and AGL in rat plasma after the plasma protein was precipitated with methanol. HPLC analysis was performed on a C18 column with UV detection at 350 nm and a mobile phase of methanol-0.2% phosphoric acid (1 : 1, v/v). Calibration curves of LGL and AGL were linear over the concentration range of 0.16-20.0 and 0.06-7.20 µg mL⁻¹, respectively. The accuracy and precision of the two analytes at low, medium and high concentrations were within the range of -3.4% to 8.1%. The relative standard deviations (RSDs) of the intra- and inter-day precisions were less than 11.7% and 10.0%, respectively. The extraction recoveries (n = 5) varied from 91.9% to 104.1% for LGL and from 92.6% to 109.3% for AGL. The method was fully validated and successfully applied to a pharmacokinetic study of LGL and AGL in rat plasma after the intravenous administration of HSE.

The exposure of luteolin is much lower than that of apigenin in oral administration of Flos Chrysanthemi extract to rats.

Luteolin (3',4',5,7-tetrahydroxyflavone) and apigenin (4',5,7-trihydroxyflavone) are two common flavones and major bioactive components in Flos Chrysanthemi extract (FCE). Although FCE contains approximately equal amounts of luteolin (6.5%, w/w) and apigenin (5.4%, w/w), luteolin showed a much lower exposure than apigenin when FCE was orally administered to rats. The aim of the present study is to elucidate the mechanisms that caused the pharmacokinetic difference between luteolin and apigenin in rats. The results of an in situ rat intestinal single-pass perfusion model showed that the permeability of luteolin (k(a), 7.96×10⁻² min⁻¹ and P(eff), 4.87×10⁻³ cm/min) was about 50% that of apigenin (k(a), 18.5×10⁻² min⁻¹ and P(eff), 10.8×10⁻³ cm/min), which agreed with the observation that oral bioavailability of luteolin (30.4%) from FCE was significantly lower than that of apigenin (51.1%). On the other hand, luteolin was much more unstable than apigenin during the incubation with primary rat hepatocytes, and methylated metabolites of luteolin were detected after incubation. In addition, further metabolism of methylated luteolin also contributed to the faster elimination of luteolin. In conclusion, luteolin and apigenin are very similar in structure, however, one-hydroxyl difference gives them different characteristics in absorption and metabolism, which results in much lower exposure of luteolin than apigenin when FCE is orally administered to rats.

Development of an LC-MS method for simultaneous quantitation of amentoflavone and biapigenin, the minor and major biflavones from Hypericum perforatum L., in human plasma and its application to real blood.

INTRODUCTION: Biflavones of Hypericum perforatum L. are bioactive compounds used in the treatment of inflammation and depression. Determination of amentoflavone and biapigenin from blood is challenging owing to their similar structures and low concentrations. OBJECTIVE: To develop a rapid, sensitive and accurate method based on liquid-phase extraction followed by high-performance liquid chromatography and electrospray ionisation mass spectrometry (HPLC-ESI-MS) for quantification of biflavones in human plasma. METHODOLOGY: After extraction from blood, the analytes were subjected to HPLC with an XTerra® MS C(18) column and a binary mobile phase consisting of 2% formic acid in water and acetonitrile under isocratic elution conditions, with ESI-MS detection in the negative ion mode and multiple reaction monitoring (MRM). RESULTS: Both calibration curves showed good linearity within the concentration range 1-500 ng/mL. Limits of detection (S/N = 3) were 0.1 ng for pure substances and the limits of quantitation (S/N = 5) were 1.0 ng/mL from analyte-spiked serum. The grand mean recovery was 90% from several subsamples of each biflavone. The imprecision (RSD) of peak areas was between 5% (intraday) and 10% (interday) for high concentrations (250 ng/mL) and between 10% (intraday) and 15% (interday) for low concentrations (1 ng/mL). Inaccuracy of the mean was less than 20% at the lower limit of quantitation. CONCLUSION: The developed and validated method for determination of biflavones from human plasma was effectively applied to pharmacokinetic studies of 13 probands and preliminary results indicate biphasic concentration-time curves.

[Intestinal lymphatic transport of breviscapine orally administered in rat].

Double cannulation model of conscious rat allowing simultaneous collection of mesenteric lymph and jugular venous blood was established to investigate the intestinal lymphatic transport of breviscapine orally administered in rat. The concentrations of breviscapine in plasma and lymph were determined by HPLC. The pharmacokinetics of breviscapine after oral and intravenous administration was evaluated in the conscious rat model. It was observed that scutellarin distributed from blood circulation to lymphatic system after intravenous injection. The cumulative lymphatic transport amount within 12 h was (2.78 +/- 0.25) microg, equivalent to 0.0792% of intravenous dose. After oral administration of scutellarin to double-cannulation rats, the cumulative lymphatic transport amount within 12 h was (0.92 +/- 0.08) microg, equal to 0.0083% of oral dose. The absolute bioavailability of breviscapine orally administered to double-cannulation rats was 4.91%, indicating that scutellarin was mainly absorbed into the bloodstream through the portal vein. Lymphatic transport of scutellarin appears to reflect high affinity for the lymph lipoproteins to chylomicron. This study provided a biopharmaceutics basis for developing oral lipid delivery system for the promotion of intestinal lymphatic transport to improve oral bioavailability of breviscapine.