Development and Validation of a UPLC-MS/MS Method for the Quantitative Determination and Pharmacokinetic Analysis of Cirsimarin in Rat Plasma.

Cirsimarin is a bioactive antilipogenic flavonoid isolated from the cotyledons of Abrus precatorius and represents one of the most abundant flavonoids present in this plant species. Cirsimarin exhibits excellent antioxidant, lipolysis, and other biological properties; it can effectively trigger lipid movement and demonstrates antiobesity effects. In this work, an ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of cirsimarin in rat plasma after intravenous administration. A standard curve of cirsimarin in blank rat plasma was generated over the concentration range of 1-3000 ng/mL. Six rats were administered cirsimarin intravenously (1 mg/kg). The method only required 50 µL of plasma for sample preparation, and the plasma proteins were precipitated with acetonitrile to pretreat the plasma sample. The precisions of cirsimarin in rat plasma were less than 14%, while the accuracies varied between 92.5% and 107.3%. In addition, the matrix effect varied between 103.6% and 107.4%, while the recoveries were greater than 84.2%. This UPLC-MS/MS method was then applied in measuring the pharmacokinetics of cirsimarin in rats. The AUC(0-t) values of cirsimarin from the pharmacokinetic analysis were 1068.2 ± 359.2 ng/mL·h for intravenous administration. The half-life (t 1/2) was 1.1 ± 0.4 h (intravenous), indicating that the metabolism of the compound was quick in the rats. Exploring the pharmacokinetics of cirsimarin in vivo can help better understand its metabolism.

Simple and fast UPLC-MS method for quantifying the anti-inflammatory candidate 5'-methoxynobiletin in rat plasma: Validation and application in a preliminary pharmacokinetic study.

This study presents the development and validation of a fast and simple bioanalytical ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method intended for quantifying the anti-inflammatory candidate 5'-methoxynobiletin (5'-MeONB) in rat plasma. Standard of 5'-MeONB was purified from A. conyzoides extract by using preparative HPLC. After a pretreatment of plasma samples with acetonitrile, chromatographic separations were efficiently achieved with a C18 column using a 9 min gradient system of 0.1% aqueous formic acid and acetonitrile as eluent. Drug candidate 5'-MeONB and chrysin (internal standard, IS) detection were carried out using ESI+ through the extracted ion chromatograms approach, monitored at m/z 433.1494 (for 5'-MeONB, tR:1.78 min) and m/z 255.0657 (for IS, tR:1.57 min). Method was validated according to US FDA guidelines, presenting linearity (R2 > 0.999) over concentration range of 30-750 ng/mL. Relative standard deviation (RSD) of repeatability and intermediary precision respectively ranged between 1.93-3.65% and 2.16-7.54%, considering lower limit of quantitation (30 ng/mL) and quality control (90, 360 and 600 ng/mL) samples, while accuracy was between 82.51 and 109.44%. Moreover, no interference from plasma endogenous substances, no carryover effect, and no influence of extraction method even in hemolyzed blood samples were observed. Sample stability in auto-sampler and long-term -80 °C storage, as well as matrix effect were within acceptable limits. For the first time, using the validated UPLC-MS bioanalytical method, the plasma pharmacokinetics of 5'-MeONB following 2 mg/kg intravenous bolus dosing to Wistar rats was characterized allowing the determination of the parameters describing drug distribution and elimination.

Comparative Analyses of Bioavailability, Biotransformation, and Excretion of Nobiletin in Lean and Obese Rats.

Nobiletin, one of the prevalent polymethoxyflavones in citrus peels, was reported to possess various health benefits. We conducted the excretion study and pharmacokinetics study of nobiletin via oral administration and intravenous injection and 15 day consecutive dosing study using the high fat diet-induced obese rats and their lean counterparts. By comparing the demethylated metabolite profiles in the urine and feces, gut microbiota demonstrated greater biotransformation activity on nobiletin than the host. The absolute oral bioavailability of nobiletin in lean (22.37% ± 4.52%) and obese (18.67% ± 4.80%) rats has a negligible statistically significant difference (P > 0.05). However, a higher extent of demethylated metabolites was found in the feces and plasma of obese rats than lean rats (P < 0.05). Moreover, the consecutive dosing of nobiletin might lead to a higher extent of demethylated metabolites in the plasma and in feces. These results suggested that gut microbiota played important roles in nobiletin metabolism.

Simultaneous characterization of multiple Psoraleae Fructus bioactive compounds in rat plasma by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry for application in sex-related differences in pharmacokinetics.

A method for the simultaneous quantification of 13 bioactive compounds (psoralen, isopsoralen, isobavachin, bakuchalcone, neobabaisoflavone, bavachin, corylin, psoralidin, isobavachalcone, bavachinin, corylifol A, bavachalcone, and bakuchiol) by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry has been developed and validated in rat plasma. Osthol was used as an internal standard and plasma samples were pretreated with one-step liquid-liquid extraction. These analytes were separated using a gradient mobile phase system of water and acetonitrile at a flow rate of 0.2 mL/min on a reverse-phase C18 column and analyzed in the selected multiple reactions monitoring mode. All calibration curves were linear (r > 0.9952) over the tested ranges. The intra- and interday accuracy and precisions of these analytes at three different concentration levels were within the acceptable limits of <15% at all concentrations. The mean recoveries of these analytes at three concentrations were more than 60.2% and the matrix effects were in the range of 85-115%. Stability studies proved that the analytes were stable under the tested conditions. The developed method was applied to evaluating the pharmacokinetic study of 13 bioactive compounds after oral administration of Psoraleae Fructus in rat of different genders. Some active compounds in Psoraleae Fructus had sex-related pharmacokinetics.

Determination of isosinensetin in rat plasma by UHPLC-MS/MS: Application to oral and intravenous pharmacokinetic study in healthy rats.

Isosinensetin is a polymethoxyflavone existing in various kinds of citrus. It has exhibited significant anti-proliferative activity and herb-drug interaction. To date, a specific determination method to quantify isosinensetin concentration in biological matrix has not been developed. In the present study, a highly specific, simple and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) approach was developed and validated for quantification of isosinensetin in rat plasma with subsequent application to a pharmacokinetic study. Isosinensetin and lysionotin (internal standard, IS) were extracted from rat plasma by a single step protein precipitation using acetonitrile as precipitation agent. The chromatographic separation was conducted using an Agilent C18 column with a gradient elution system (0.1 % formic acid aqueous solution and acetonitrile) within 3.5 min. An electrospray ionization (ESI) source operating in positive mode and multiple reaction monitoring (MRM) were used to monitor the transitions of m/z 373.1 → 343.1 for isosinensetin and m/z 345.1 → 315.1 for IS. The developed method was linear within the range of 1-1000 ng/mL and fully validated according to FDA guidelines. The accuracy values reported as relative errors were between 2.0 and 10.0 % for three quality control levels (2, 400 and 800 ng/mL) and lower limit of quantification (LLOQ). The precisions were ≤11.1 % for quality controls and ≤18.1 % for LLOQ. The recoveries and matrix effects of isosinensetin were in the range of 83.4-87.7 % and 105.6-108.8 %, respectively. Other parameters such as selectivity, carryover effect, dilution integrity and stability were also validated and met the acceptance criteria. The method was applied to a pharmacokinetic study in rats following oral and intravenous administration of isosinensetin. Isosinensetin was rapidly absorbed with a poor bioavailability of 2.19 % and quickly eliminated with mean half-life of 1.40 h and 1.76 h for oral and intravenous route, respectively.

Quantitative determination of characteristic components from compound of Lysionotus pauciflorus Maxim. by LC-MS/MS and its application to a pharmacokinetic study.

Tuberculosis of cervical lymph nodes is called scrofula in Traditional Chinese Medicine (TCM). Clinical manifestation is that unilateral or bilateral neck can have multiple enlarged lymph nodes of different sizes. Current therapeutic drugs include Lysionotus pauciflorus Maxim. tablets and compound of Lysionotus pauciflorus Maxim., which have a significant effect on tuberculosis of cervical lymph nodes. This compound is composed of three herbs, Lysionotus pauciflorus Maxim., Prunella vulgaris L. and Artemisia argyi Levl.et Vant. A selective and sensitive LC-MS/MS method was established and validated in rat plasma for the first time. Chromatographic separation was achieved on a Wonda Cract ODS-2 C18 Column (150 mm × 4.6 mm, 5 µm). The mobile phase contained 0.1% formic acid aqueous solution and acetonitrile with a flow rate of 0.8 mL/min. The detection was performed in negative electrospray ionization mode and the precursor/product ion transitions of six components and internal standard (IS) sulfamethoxazole were quantified in multiple reaction monitoring (MRM) using QTRAP-3200 MS/MS. The method fulfilled US Food and Drug Administration guidelines for selectivity, sensitivity, accuracy, precision, matrix effect, extraction recovery, dilution integrity, and stability. This proposed method was then successfully applied to a pharmacokinetic study after oral administration of 10 mL/kg compound extracts in rats. The pharmacokinetic parameters and plasma concentration-time profiles would prove valuable in pre-clinical and clinical investigations on the disposition of compound medicine.

Pharmacokinetic study of eight bioactive components following oral administration of Zhiqiao Gancao decoction and observation of its clinical efficacy.

Zhiqiao Gancao (ZQGC) decoction is widely used in China due to its therapeutic effect on lumbar disc herniation (LDH). In this study, we compared the clinical therapeutic effects among oral ZQGC decoction treatment, bed rest, and oral anti-inflammatory drug celecoxib treatment using visual analog scale, Oswestry Disability Index, and MacNab scores. The results showed that ZQGC decoction can significantly improve the symptoms of patients with LDH. A selective, sensitive, and rapid ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of eight bioactive components in rat plasma. The plasma samples were extracted by simple protein precipitation with methanol. The protonated analytes were quantitated simultaneously in positive and negative ion modes by multiple reaction monitoring with a mass spectrometer. The calibration curve of eight components in plasma showed good linearity (r > .996) and the extraction recovery was 81.19% ± 2.15% - 100.39 ± 3.36 (relative standard deviation: 1.21%-10.70%). The accuracy of all the lower limit of quantitation values was quantified within 80%-120%, and the precision was less than 15%. This validated method was successfully applied to the pharmacokinetics study in rat plasma after ZQGC decoction oral treatment. Our research can provide experimental basis for the rational clinical application of ZQGC decoction in the treatment of LDH.

Glucose Tolerance Test and Pharmacokinetic Study of Kaempferia parviflora Extract in Healthy Subjects.

Kaempferia parviflora Wall. ex Baker (KP), Krachaidam in Thai or Thai ginseng, is a herbal medicine that has many potential pharmacological effects. The effect of KP extract on blood glucose level in rodent was reported. This study focused on the oral glucose tolerance test and pharmacokinetic study in healthy volunteers administered with KP extract (90 and 180 mg/day, placebo). The oral glucose tolerance tests were performed at baselines and 28-days of administration. The pharmacokinetics were determined after a single dose administration of the tested products using 3,5,7,3',4'-pentamethoxyflavone (PMF) and 5,7,4'-trimethoxylflavone (TMF) as markers. The results showed that glucose metabolism via oral glucose tolerance test was not affected by KP extract. Blood glucose levels of volunteers at 120 min after glucose loading were able to be returned to initial levels in placebo, KP 90 mg/day, and KP 180 mg/day groups both at baseline and 28-days of administration. The results of the pharmacokinetic study revealed that only TMF and PMF, but not 5,7-dimethoxyflavone (DMF) levels could be detected in human blood. The given doses of KP extract at 90 and 180 mg/day showed a linear dose-relationship of blood PMF concentration whereas blood TMF was detected only at high given dose (180 mg/day). The half-lives of PMF and TMF were 2-3 h. The maximum concentration (Cmax), area under the curve of blood concentration and time (AUC), and time to maximum concentration (Tmax) values of PMF and TMF estimated for the 180 mg/day dose were 71.2 ± 11.3, 63.0 ± 18.0 ng/mL; 291.9 ± 48.2, 412.2 ± 203.7 ng∙h/mL; and 4.02 ± 0.37, 6.03 ± 0.96 h, respectively. PMF was quickly eliminated with higher Ke and Cl than TMF at the dose of 180 mg/day of KP extract. In conclusion, the results demonstrated that KP extract had no effect on the glucose tolerance test. In addition, this is the first demonstration of the pharmacokinetic parameters of methoxyflavones of KP extract in healthy volunteers. The data suggest the safety of the KP extract and will be of benefit for further clinical trials using KP extract as food and sport supplements as well as a drug in health product development.

An HPLC-MS/MS method for the simultaneous determination of luteolin and its major metabolites in rat plasma and its application to a pharmacokinetic study.

A selective and accurate HPLC-MS/MS method was established to simultaneously quantify luteolin and its active metabolites (diosmetin, chryseriol, and luteolin-7-O-glucuronide) in rat plasma. The analytes were separated on a C18 column with a mobile phase of water containing 0.5% formic acid and acetonitrile under gradient elution to shorten the total chromatographic run time and increase the resolution of diosmetin and chryseriol. A triple quadruple mass spectrometer coupled with an electrospray ionization source in the negative ion mode was used to detect the analytes. The multiple reaction monitoring transitions were of m/z 284.9→132.9 for luteolin, m/z 298.9→283.9 for diosmetin and chryseriol, m/z 461.1→284.9 for luteolin-7-O-glucuronide, and m/z 300.9→150.9 for the internal standard. The method was linear within the concentration ranges of 0.06-90 µg/mL for luteolin, 0.03-12 µg/mL for diosmetin, 0.015-4.8 µg/mL for chryseriol, and 0.06-60 µg/mL for luteolin-7-O-glucuronide. The intra- and interday precisions were all within 6.0%. Accuracy ranged from -3.2 to 6.4%. The matrix effect and instability were not observed during bioanalysis. This method was used to study the pharmacokinetic characteristics of luteolin and its metabolites in rats after treatment with luteolin.

A target-group-change strategy based on the UPLC-Q-TOF-MSE method for the metabolites identification of Fufang-Xialian-Capsule in rat's plasma.

Fufang-Xialian-Capsule (FXL) is a traditional Chinese medicine (TCM) formula which was utilized to treat chronic atrophic gastritis. Despite the chemical constituents have been clarifying by our previous studies, but the metabolism of FXL after oral was still unclear. In order to clarify the mechanism of these absorbed components, a target-group-change (TGC) strategy was utilized to analysis the collected data. This strategy include five steps: (1) acquired the mass spectra data and tandem mass spectra data simultaneously; (2) confirmed the prototype absorbed into blood and the tandem mass behavior of prototype; (3) clarified the potential group change of prototypes after metabolism by Metabolynx XS software; (4) confirmed the target group change acquired by compare the tandem mass behavior of metabolites with their prototypes; (5) inferred the position of group change occurred and metabolic pathways of each prototypes. Based on the TGC strategy, the structure of metabolites and the metabolic pathways of FXL were confirmed. The main group change behaviors on the prototypes after metabolism include demethylation, methylation, hydroxylation and glucuronide conjugation. As the results, there were 33 metabolites transformed from 11 prototypes confirmed, these 11 prototypes include 4 flavones, 5 alkaloids and 2 ginsenosides. All the metabolites could be identified or tentatively characterized according to the structure of metabolites and previous reports.

Polyphenol Phase-II Metabolites are Detectable in Human Plasma after Ingestion of 13 C Labeled Spinach-a Pilot Intervention Trial in Young Healthy Adults.

SCOPE: After intrinsic labeling of spinach (Spinacia oleracea L., Chenopodiaceae) with 13 CO2 , we investigated if labeled polyphenol metabolites were detectable in human plasma. METHODS AND RESULT: In a pilot intervention trial, five healthy men consumed 5 g freeze-dried 13 C labeled spinach, including a total amount of 160 µmol methoxyflavonols, including 70 µmol 5,3',4'-trihydroxy-3-methoxy-6,7-methylendioxyflavone-4'-glucuronide. Plasma samples of all subjects were analyzed with regard to their 13 C/12 C ratio. Additionally, 13 C labeled metabolites of patuletin, spinacetin, and 5,3',4'-trihydroxy-3-methoxy-6,7-methylendioxyflavone (TMM) were analyzed in plasma samples in a subgroup of three subjects. TMM-glucuronide, TMM-sulfate, and spinacetin-glucuronide-sulfate, the latter as 12 C113 C16 and 13 C17 isotopologs, were tentatively identified. Plasma concentration of TMM-glucuronide and TMM-sulfate reached cmax from 19.1-54.3  and 22.5-125.5 nmol L-1 , respectively, 7-9 h post-ingestion. CONCLUSION: It seems likely that 13 C labeled TMM-glucuronide and TMM-sulfate are phase-II metabolites which were converted after colonic transformation. Variations in plasma kinetics were observed for these two metabolites and may be attributed to the individual composition of the microbiota. We conclude that 13 C labeled polyphenol metabolites are detectable and quantifiable in human plasma.

Appetite-Inducing Effects of Homoeriodictyol: Two Randomized, Cross-Over Interventions.

SCOPE: Anorexia of aging, characterized by a decrease in appetite and/or food intake, is a major risk factor of under-nutrition and adverse health outcomes in elderly people. Recent in vitro evidence suggests homoeriodictyol (HED), a naturally occurring, bitter-masking flavanone, as a promising agent to increase appetite and food intake. METHODS AND RESULTS: In two cross-over intervention trials, 30 mg NaHED, either solely (n = 10, Study I) or in combination with a 75 g glucose load (n = 17, study II) were administered to healthy adult subjects. Ratings of hunger were assessed at fasting and either 30 min (Study I) or 120 min (Study II) post intervention. Ad libitum energy intake from a standardized breakfast and plasma changes in hunger-/satiety-associated hormones PYY, GLP-1, ghrelin and serotonin were determined after blood drawings. Effects were more pronounced when NaHED was administered in combination with 75 g glucose since ad libitum energy (+ 9.52 ± 4.60%) and protein (+ 7.08 ± 7.97%) intake as well as plasma ΔAUC ghrelin values increased in study II solely, whereas plasma serotonin concentrations decreased after both interventions. CONCLUSIONS: NaHED demonstrated appetizing effects in healthy adults when administered with a glucose load. Long-term intervention studies are warranted to verify these effects in compromised subjects.

Metabolic profile study of 7, 8-dihydroxyflavone in monkey plasma using high performance liquid chromatography-tandem mass spectrometry.

7, 8-Dihydroxyflavone, as a high-affinity tropomyosin-receptor-kinase B agonist, can mimic the physiological actions of brain-derived neurotrophic factor and exert a variety of neurological actions in numerous models including Parkinsons disease, depression, learning and memory. Nonetheless, a limited number of studies have been focused on its metabolism in mammal and no methodology has been reported for the determination of 7, 8-DHF and its metabolites. Herein, we developed a rapid, sensitive and accurate method using high performance liquid chromatography-tandem mass spectroscopy for the determination of 7, 8-DHF and its metabolites in monkey plasma. The lower limits of quantification for analytes were 0.4-2.0ngmL-1. The intra-day and inter-day precisions (relative standard deviation, %) of analytes were within 11.83%, and the accuracy (relative error, %) ranged from -6.86 to 14.00%. The mean extraction recoveries for analytes were more than 89.14%. This validated method was successfully applied to the metabolic profile study of 7, 8-DHF in monkey plasma. The results indicated that 7, 8-DHF undergoes methylation, glucuronidation and/or sulfation, and the conjugated forms are the main metabolites in monkey plasma. We further demonstrated that methylated 7, 8-DHF can be also conjugated with glucuronidation/sulfation, and the methylation occurs mainly in the 8 position.

Quantitative bioanalysis of bavachalcone in rat plasma by LC-MS/MS and its application in a pharmacokinetics study.

This study aims to develop and validate a simple and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for investigating the pharmacokinetic characteristics of bavachalcone. Liquid-liquid extraction was used to prepare plasma sample. Chromatographic separation of bavachalcone and IS was achieved using a Venusil ASB C18 (2.1 × 50 mm, 5 µm) column with a mobile phase of methanol (A)-water (B) (70:30, v/v). The detection and quantification of analytes was performed in selected-reaction monitoring mode using precursor → product ion combinations of m/z 323.1 → 203.2 for bavachalcone, and m/z 373.0 → 179.0 for IS. Linear calibration plots were achieved in the range of 1-1000 ng/mL for bavachalcone (r2 > 0.99) in rat plasma. The recovery of bavachalcone ranged from 84.1 to 87.0%. The method was precise, accurate and reliable. It was fully validated and successfully applied to pharmacokinetic study of bavachalcone.

Plasma Pharmacokinetics, Bioavailability, and Tissue Distribution of Four C-Glycosyl Flavones from Mung Bean (Vigna radiata L.) Seed Extracts in Rat by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

The main polyphenols in mung bean (Vigna radiata L.) seed (MBS), an edible legume with various biological activities, are C-glycosyl flavones (vitexin, isovitexin, isovitexin-6″-O-α-l-glucoside, and dulcinoside). In our study, a validated ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantitate the concentrations of four C-glycosyl flavones from MBS extracts in the plasma and various tissues of rats and successfully applied to study their pharmacological profile and tissue distribution in vivo. Four C-glycosyl flavones were rapidly absorbed after oral administration, achieving a Cmax at around 1.5 h, and they could be distributed widely and rapidly in tested tissues. The concentrations of four C-glycosyl flavones in all of the tested tissues decreased obviously in 4 h, which indicated that there was not a trend of long-term accumulation of them. This is the first time to report on pharmacokinetic and tissue distribution studies of four C-glycosyl flavones in rat. The results provided a significative basis for the application of MBS.

Simultaneous determination of ten bioactive constituents of Sanjie Zhentong Capsule in rat plasma by ultra-high-performance liquid chromatography tandem mass spectrometry and its application to a pharmacokinetic study.

Sanjie Zhentong capsule, a well-known traditional Chinese medicine prescription, are used for the treatment of endometriosis-related diseases. In this study, a simple, rapid and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of ten bioactive constituents, including peimine, peiminine, peimisine, loureirin A, loureirin B, 7,4'-dihydroxyflavone, pterostilbene, ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1 in rat plasma after oral administration of Sanjie Zhentong capsule. The sample preparations for protein removal was accomplished using a simple methanol precipitation method. The analytes were completely separated from the endogenous compounds on an Agilent Poroshell 120 SB-C18 column (4.6mm×150mm, 2.7µm) using an isocratic elution with methanol - 0.1% formic acid aqueous (4/1, v/v) as a mobile phase. The single-run analysis time was as short as 14.0min. The inter-day and intra-day precision of the quality control samples exhibited relative standard deviations (RSD) <9.5% and the accuracy values ranged from -8.6% to 15.0%. The lower limits of quantification (LLOQ) were 10, 10, 10, 10, 10, 10, 5, 10, 10 and 20ng/mL for peimine, peiminine, peimisine, loureirin A, loureirin B, 7,4'-dihydroxyflavone, pterostilbene, ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1, respectively. The analytical method was successfully applied to a pharmacokinetic study of the multi-components after oral administration of Sanjie Zhentong Capsule in rats.

Structural Pharmacokinetics of Polymethoxylated Flavones in Rat Plasma Using HPLC-MS/MS.

The purpose of this study was to investigate the pharmacokinetics of the polymethoxylated flavonoids kumatakenin, pachypodol, and retusin, which contain two, three, or four methoxy substitutions, using a validated ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method in rats. The pharmacokinetic results demonstrated that the elimination half-lives for kumatakenin, pachypodol, and retusin were 30 ± 11.6, 39.4 ± 19.5, and 106.9 ± 26 min, respectively, for the low dose group and 54.5 ± 16.5, 33.8 ± 10, and 134.6 ± 34.7 min for the high dose group. The results suggested that the area under the curve values (AUC) for the analytes did not correlate with the number of methoxy groups. Pachypodol had the lowest AUC, which may have been correlated with lipophilicity, for both the low and high dose groups. In conclusion, the polymethoxylated flavonoid pachypodol is more hydrophilic than kumatakenin or retusin, which were correlated with the pharmacokinetic results.

Identification of absolute conversion to geraldol from fisetin and pharmacokinetics in mouse.

Fisetin (3,3',4',7-tetrahydroxyflavone) is a flavonoid found in several fruits, vegetables, nuts, and wine and has anti-oxidant, anti-inflammatory, and anti-angiogenic properties. Geraldol is the 3'-methoxylated metabolite of fisetin (3,4',7-trihydroxy-3'-methoxyflavone). The concentration of fisetin and geraldol in mouse plasma was determined by LC-MS/MS, following direct protein precipitation. These concentrations were determined after administration of fisetin at doses of 2mg/kg (i.v.) and 100 and 200mg/kg (p.o.). The method was validated in terms of linearity, accuracy, precision, matrix effect, and stability. The pharmacokinetics parameters of fisetin and geraldol were successfully determined using a validated method in mice. Results indicated that fisetin was very rapidly methylated to geraldol in vivo. Following administration of fisetin, it was observed that the Cmax and AUC values for geraldol were higher than those of fisetin. The absolute bioavailability of fisetin was calculated as 7.8% and 31.7% after oral administration of 100 and 200mg/kg fisetin, respectively. This method was successfully applied to determine the pharmacokinetic parameters of fisetin and its main metabolite geraldol in mouse plasma. Geraldol was the dominant circulating metabolite after fisetin administration in vivo.

Synthesis of a highly water-soluble acacetin prodrug for treating experimental atrial fibrillation in beagle dogs.

We previously reported that duodenal administration of the natural flavone acacetin can effectively prevent the induction of experimental atrial fibrillation (AF) in canines; however, it may not be used intravenously to terminate AF due to its poor water-solubility. The present study was to design a water-soluble prodrug of acacetin and investigate its anti-AF effect in beagle dogs. Acacetin prodrug was synthesized by a three-step procedure. Aqueous solubility, bioconversion and anti-AF efficacy of acacetin prodrug were determined with different methodologies. Our results demonstrated that the synthesized phosphate sodium salt of acacetin prodrug had a remarkable increase of aqueous solubility in H2O and clinically acceptable solution (5% glucose or 0.9% NaCl). The acacetin prodrug was effectively converted into acacetin in ex vivo rat plasma and liver microsome, and in vivo beagle dogs. Intravenous infusion of acacetin prodrug (3, 6 and 12 mg/kg) terminated experimental AF without increasing ECG QTc interval in beagle dogs. The intravenous LD50 of acacetin prodrug was 721 mg/kg in mice. Our preclinical study indicates that the synthesized acacetin prodrug is highly water-soluble and safe; it effectively terminates experimental AF in beagle dogs and therefore may be a promising drug candidate for clinical trial to treat patients with acute AF.

Enhanced Oral Bioavailability of Pueraria Flavones by a Novel Solid Self-microemulsifying Drug Delivery System (SMEDDS) Dropping Pills.

To improve bioavailability of pueraria flavones (PF), a self-microemulsifying drug delivery system (SMEDDS) dropping pills composed of PF, Crodamol GTCC, Maisine 35-1, Cremophor RH 40, 1,2-propylene glycol and polyethylene glycol 6000 (PEG6000) was developed. Particle size, zeta potential, morphology and in vitro drug release were investigated, respectively. Pharmacokinetics, bioavailability of PF-SMEDDS dropping pills and commercial Yufengningxin dropping pills were also evaluated and compared in rats. Puerarin treated as the representative component of PF was analyzed. Dynamic light scattering showed the ability of PF-SMEDDS dropping pills to form a nanoemulsion droplet size in aqueous media. The type of media showed no significant effects on the release rate of PF. PF-SMEDDS dropping pills were able to improve the in vitro release rate of PF, and the in vitro release of these dropping pills was significantly faster than that of Yufengningxin dropping pills. There was a dramatic difference between the mean value of t1/2, peak concentration (Cmax), the area of concentration-time curve from 0 to 6 h (AUC0-6 h) of PF-SMEDDS dropping pills and that of commercial Yufengningxin dropping pills. A pharmacokinetic study showed that the bioavailability of PF was greatly enhanced by PF-SMEDDS dropping pills. The value of Cmax and relative bioavailability of PF-SMEDDS dropping pills were dramatically improved by an average of 1.69- and 2.36-fold compared with that of Yufengningxin dropping pills after gavage administration, respectively. It was concluded that bioavailability of PF was greatly improved and that PF-SMEDDS dropping pills might be an encouraging strategy to enhance the oral bioavailability of PF.