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.

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.

DPPH·-luminol chemiluminescence system and its application in the determination of scutellarin in pharmaceutical injections and rat plasma with flow injection analysis.

In this article, a DPPH·-luminol chemiluminescence (CL) system was reported and the CL mechanism was discussed according to the CL kinetic properties after sequence injecting DPPH· into the DPPH·-luminol reaction mixture. It was observed that scutellarin could inhibit the CL response of the DPPH·-luminol system. Based on this observation, a simple and rapid flow injection CL method was developed for the determination of scutellarin using the inhibition effect in alkaline medium. The optimized chemical conditions for the CL reaction were 5 × 10-6  mol/L DPPH· and 1.0 × 10-4  mol/L luminol in 0.01 mol/L NaOH. Under optimized conditions, the CL intensity was inversely proportional to the concentration of scutellarin over the ranges 5-2000 and 40-3200 ng/ml in pharmaceutical injection and rat plasma, respectively. The limits of detection (S/N = 3) were 5 and 40 ng/ml in preparations and rat plasma, respectively. Furthermore, the precision, recovery and stability of the validated method were acceptable for the determination of scutellarin in both pharmaceutical injections and rat plasma. The presented method was successfully applied in the determination of scutellarin in pharmaceutical injections and real rat plasma samples.

Hepatic, gastric and intestinal first-pass effects of vitexin-2''-O-rhamnoside in rats by ultra-high-performance liquid chromatography.

Previous research in our laboratory found that the absolute bioavailability of vitexin-2''-O-rhamnoside (VR) was quite low at 4.89%. A rapid and sensitive UHPLC method using hesperidin as an internal standard was therefore developed and validated to investigate the reasons for this by determining VR in rat plasma after administering intravenously, intraportally (5 mg/kg), intraduodenally and intragastrically (40 mg/kg) to the rat model of the hepatic, gastric and intestinal first-pass effects. As only a high intestinal first-pass effect of VR was found, that is, there existed a low bioavailability of VR (2.40%), inhibitors of P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A), including verapamil, cyclosporin A and midazolam, and absorption enhancers, including bile salts and borneol, combined with VR, were instilled into duodenum to evaluate the effects on bioavailability of VR. The results demonstrated that area under the concentration-time curve (AUC) values of VR slightly increased after administration of verapamil, cyclosporin A and midazolam, indicating that CYP3A and P-gp do not play an important role in the first-pass effect in the intestine. AUC values of VR significantly increased after administering bile salts or borneol, indicating that the low bioavailability of VR was mainly related to its poor absorption in the intestine.

Development and validation of HPLC method for vicenin-1 isolated from fenugreek seeds in rat plasma: application to pharmacokinetic, tissue distribution and excretion studies.

CONTEXT: Vicenin-1, a flavonol glycoside, has potent platelet aggregation inhibition, antioxidant, radioprotectants and anti-inflammatory activities. OBJECTIVE: To establish a rapid, simple, precise and sensitive high-performance liquid chromatography (HPLC) method for determination of vicenin-1 in rat plasma, and to investigate the pharmacokinetics, tissue distribution and excretion after a single 60 mg/kg oral dose in rats. MATERIALS AND METHODS: Vicenin-1 was extracted by solid-liquid extraction through Tulsicon® ADS-400 (0.40-1.2 mm). Chromatographic separation was achieved by HPLC with a C18 column with a mobile phase composed of water and acetonitrile (75:25 v/v) and a flow rate of 1 mL/min along with UV detection at 210 nm. RESULTS: Good linearity of calibration curve was found between 10.5 and 100.5 µg/mL (R2 = 0.995) for plasma and tissue, whereas 2.5-500 µg/mL (R2 = 0.999) for the urine and stool samples. The extraction recoveries were 98.51-99.58% for vicenin-1 in plasma, whereas intra-day and inter-day precision were validated by relative standard deviation (%RSD), that came in the ranges of 1.16-1.79% and 1.28-1.73%, respectively. The pharmacokinetics results showed Cmax (7.039 µg/mL) and Tmax (2 h) after oral administration of vicenin-1. Tissue distribution study showed that the highest concentration of vicenin-1 was achieved in the liver followed by the lung. Approximately 24.2% of its administered dose was excreted via urinary excretion route. CONCLUSION: The first-pass metabolism, poor solubility and presence of reducing sugar moiety in vicenin-1 may decrease its bioavailability. The developed method is sensitive, specific and was successfully applied to the pharmacokinetics, tissue distribution and excretion studies of vicenin-1 in rats.

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.

An enzyme-linked immunosorbent assay for the measurement of plasma flavonoids in mice fed apigenin-C-glycoside.

BACKGROUND: In the Chenopodiaceae family, the apigenin flavonoids vitexin-2-O-xyloside (VOX) and vitexin-2-O-rhamnoside (VOR) are important chemopreventive components. To investigate their bioavailability in in vivo animal studies an enzyme-linked immunosorbent assay (ELISA) method has been developed. RESULTS: The ELISA was based on polyclonal antibodies elicited in mice by injecting, as an immunogen, 4',6″-O-biapigenin (hinokiflavone, HF) conjugated to bovine serum albumin (BSA-HF). A second immunogen was synthesised by coupling an equimolar mixture of VOX and VOR to BSA (BSA-F1). The BSA-HF elicited a significant antibody response, due to 17 HF hapten groups, coupled to each BSA molecule, whereas BSA-F1 provided a very low antigenicity in respect to control animals. Antiserum raised against BSA-HF showed an antibody titre of 1:1600. Antibodies were found to be specific for the flavonols. Our results show that VOX and its metabolic products reached the concentration of 3.42 ± 0.72 µg mL⁻¹ in plasma of VOX fed animals, at the net of the control value. CONCLUSIONS: By using the ELISA, the concentration of apigenin flavonoids and their metabolites can be detected in VOX- or VOR-supplemented animals. The assay represents a useful tool for rapid screening to compare bioavailability of apigenin flavonoids in respect to control animals.

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).

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.

[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.

Absorption and disposition of scutellarin in rats: a pharmacokinetic explanation for the high exposure of its isomeric metabolite.

Scutellarin or scutellarein-7-O-glucuronide (S-7-G) is a flavonoid used in the treatment of cardiovascular diseases. After oral administration to humans, S-7-G can hardly be detected, whereas its isomeric metabolite [scutellarein-6-O-glucuronide (S-6-G)] dominates in plasma. A preliminary study in rats also revealed a low bioavailability of S-7-G, as well as a high plasma concentration of S-6-G. Therefore, the present study tried to explore the possible causes of the unusual pharmacokinetics of scutellarin in humans through investigating the absorption and disposition of S-7-G in rats. After oral administration to rats, S-7-G was largely hydrolyzed in the intestinal tract and was absorbed as aglycone. While passing through the intestinal wall, aglycone was extensively glucuronidated into S-7-G and S-6-G (approximately 20:1), which subsequently entered the mesenteric blood (approximately 15:1). However, because S-7-G exhibited more rapid uptake in hepatocytes, was glucuronidated at a 2.7-fold higher rate in the liver, and was excreted in greater amounts through bile and urine than S-6-G, the S-7-G/S-6-G ratio eventually declined to approximately 1.5:1 in the systemic circulation. Findings revealed that S-7-G cannot be absorbed directly; S-7-G and S-6-G in the body were mostly generated from aglycone in the intestinal wall; a larger amount of S-7-G than S-6-G entered the mesenteric blood at the absorption stage, but the gap between them shrank quickly mainly because of the higher hepatic first-pass elimination of S-7-G. These findings in rats are of great value as reference for further study to accurately interpret the pharmacokinetics of S-7-G in humans.

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.

[Effect of pravastatin on transportation of scutellarin in mouse liver and its mechanism].

This study is to investigate the transportation of scutellarin in cell and live models and study on mechanism of absorption and transport of scutellarin in mouse liver. The concentration of scutellarin in plasma and liver from control and pretreated groups was determined by high performance liquid chromatography. The uptake of scutellarin was examined in control hepatocytes group, induced hepatocytes group and induced hepatocytes plus pravastatin group. Pravastatin can affect the pharmacokinetics of scutellarin in mouse: CL is decreased while AUC is increased. The scutellarin absorption of hepatocyte induced group was higher than that of control group, but was decreased in the group with pravastatin added. The research showed that there was potential drug interaction between pravastatin and scutellarin. The drugs may compete for oatp2 mediated transport pathway consisted in the uptake of scutellarin in liver.

LC-MS/MS Determination of Apigenin in Rat Plasma and Application to Pharmacokinetic Study.

BACKGROUND: Apigenin, a natural plant flavone, has been shown to possess a variety of biological properties. OBJECTIVE: In this report, a highly selective and sensitive LC-MS/MS method was developed and validated for the determination of apigenin in rat plasma. METHODS: Analysts were separated on the HSS T3 column (1.8 µm 2.1×100 mm) using acetonitrile and 0.1% formic acid in 2mM ammonium acetate buffer at a supply rate of 0.200 mL/min as eluent in gradient model. RESULTS: Plasma samples were treated by protein precipitation using acetonitrile for the recovery ranging from 86.5% to 90.1% for apigenin. The calibration curves followed linearity in the concentration range of 0.50-500ng/mL. The inter-day and intra-day precisions at different QC levels within 13.1% and the accuracies ranged from -10.6% to 8.6%. CONCLUSION: The assay has been successfully applied to the pharmacokinetic study of apigenin in rats.

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.

The relationship between fasting plasma concentrations of selected flavonoids and their ordinary dietary intake.

Epidemiological studies suggest that a diet high in flavonoids protects against chronic diseases such as CVD and cancer. The objective of the present study was to evaluate the relationship between the intake of quercetin, kaempferol, isorhamnetin, apigenin and luteolin and their corresponding plasma concentrations, and further to explore whether these flavonoids can serve as biomarkers of their intake. Flavonoid intake and their plasma concentrations were analysed in ninety-two subjects consuming their habitual diet. Flavonoid intake was estimated with 7-d dietary records using available data on the flavonoid content of food. Plasma flavonoid concentrations were quantified by HPLC. In addition, we undertook a dietary intervention study to investigate plasma apigenin concentration after the consumption of celery leaf. The mean intake estimates of quercetin, kaempferol, isorhamnetin, apigenin and luteolin amounted to 13.58, 14.97, 12.31, 4.23 and 8.08 mg/d, respectively. The corresponding mean plasma concentrations were 80.23, 57.86, 39.94, 10.62 and 99.90 nmol/l. The mean 7 d intake of five flavonoids was positively correlated to their corresponding plasma concentrations, with correlation coefficients ranging from 0.33 to 0.51 (P < 0.05). In the dietary intervention study, the plasma apigenin concentration rose after celery leaf ingestion, and fell within 28 h to below the limit of detection (2.32 nmol/l). The present results suggest that quercetin, kaempferol, isorhamnetin, apigenin and luteolin are bioavailable from the diet. The levels of fasting plasma flavonoids seem to be suitable biomarkers of short-term intake. The combination of plasma flavonoids with their intake may prove useful when the possible health-protective effects of flavonoids are studied.

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.

UHPLC assisted simultaneous separation of apigenin and prednisolone and its application in the pharmacokinetics of apigenin.

A new ultra-high performance liquid chromatography-mass spectrometry-mass spectrometry (UHPLC-MS/MS) system has been formulated for the resolution of closely related drugs apigenin (API, a bioflavinoid) and prednisolone (PRD) from their mixture. This developed method comprised of a "BEH™ C18 column (50 mm × 2.1 mm, 1.7 µm)" using acetonitrile and 0.1% formic acid (35:65 v/v) at a supply rate of 0.25 mL·min-1 as eluent. It was found that selected eluent provided short run time (≤2.5 min) as well as better peak symmetry. Satisfactory values of chromatographic parameters such as resolution (Rs = 2.5), capacity factor (k; 13.6 and 23.4 for API and PRD respectively, selectivity (α = 1.72) and number of theoretical plates (N; 3789 and 42,435 for API and PRD respectively) indicate the efficiency of the developed method. The obtained separation was then exploited for the detection and measurement of API in rat plasma sample by means of PRD as an "internal standard" (IS). The eluted compounds in plasma were identified by tandem mass spectrometry by means of tandem quadrupole (TQ) detector ("Waters Corp., Milford, MA") fortified with an "electrospray ionisation (ESI)" source functioning in positive ionization mode. The determination of API in plasma was accomplished by means of "multiple reactions monitoring (MRM)" mode. Assortment of "ionization pairs" (m/z) was displayed in the following manner: API: 270.99 → 152.9 ("cone voltage" 57 V, "collision energy" 34 V), PRD: 403.172 → 385.224 ("cone voltage" 42 V, "collision energy" 13 V). The calibration curves followed linearity in concentration range of 05-1000 ng mL-1 with limit of detection "LOD" and limit of quantification "LOQ" of 7.30 and 22.77 ng mL-1, respectively. The developed method was validated taking into consideration various test conditions and satisfactory values of various parameters such as linearity (r2 ±â€¯SD = 0.9995 ±â€¯0.0005), interday accuracy (88-120%), interday precision % RSD = 3.30-13.65% whereas intraday accuracy (91-118%) intraday precision % RSD = 1.18-5.83) indicated its validity. The validation outcomes fulfilled the standards of united states food and drug administration "USFDA" in addition Scientific Working Group for Forensic Toxicology "SWGTOX" guiding principles and were not beyond the tolerable constraint. The process developed in plasma was efficaciously harnessed in the pharmacokinetic investigation of various formulations of API after oral administration in rats.