Studies On Quality Control And Pharmacokinetics Of Traditional Chinese Medicine Verbena Officinalis L.

Verbena Officinalis L., which is known as Mabiancao and mostly grows in South China, is a famous traditional Chinese medicine (TCM) and widely used for clearing away heat and detoxicating, promoting blood circulation and removing blood stasis inducing diuresis and excreting dampness based on the Chinese medical theory. It also can be used in folk medicine as a diuretic, expectorant and anti-rheumatic. In Navarra, Spain, it is used extensively in traditional medicine mainly because of its anti-inflammatory topical applications. In 1985, it was the first time to report Verbena Officinalis L. had antitussive effect which was associated with the components including verbenalin by Gui Chenghui in China. However, as we know, one or several categories of ingredients contribute to the pharmacodyamic material basis in most traditional Chinese medicine. Although verbenalin is a main component in Verbena Officinalis L., other compounds similar to it or some parts else exist extensively. In the past, there have been some preliminary researches about pharmacodyamic material basis. In this paper, quality control and pharmacokinetics of Verbena Officinalis L. were investigated in order to provide scientific evidence for the establishment of suitable quality control standard. The current research work provided a beneficial exploration for the Verbena Officinalis L..In this paper, as we known its pharmacodyamic material basis, A HPLC fingerprint of the total extract from Verbena Officinalis L. was established and was used for quality control. The study of main components contents and absorption in vivo is a good way for us to explain and predict a variety of events related to the efficacy and toxicity of TCM.Part one Study on the fingerprints of Verbena officinalis L. from different Sources with HPLC-PDA Objective: To establish HPLC-PDA fingerprints of Verbena officinalis L. from different areas, to provide a new method for scientific evaluation and quality control of them.Methods: The chromatographic procedure was carried out with DiamonsilTM C18 (250 mm×4.6 mm, 5μm) as an analytic column and a mixture consisting of acetonitrile and 0.05% phosphoric acid in gradient as mobile phase, the temperature of column was 30?C. The detection wavelength was set at 265 nm and the flow rate was 1.0 mL/min.Results: The mutual mode of PDA-HPLC fingerprints was set up, and the 20 common peaks were pinpointed. The similar degrees to the 12 batches Verbena officinalis L. of different producing areas were compared. The similarities in 8 batches of Verbena officinalis L. samples were all above 0.9 and those of 4 batches were between 0.8 and 0.9.Conclusion: This method of the operation is simple, quick, accurate and can be used for the identification and quality control of Verbena officinalis L.Part two Simultaneous determination of main glycosides and three flavone components in Verbena officinalis L.Objective: To develop a high performance liquid chromatography coupled with variable wavelength detector method for simultaneous determination of verbenalin and hastatoside in Verbena officinalis L.; to establish a high performance liquid chromatography coupled for simultaneous determination of 3 flavone components (luteolin; apigenin; kaempferol) in Verbena officinalis L..Methods: The dried powders of Verbena officinalis L. samples (0.5 g, 40 mesh) were accurately weighed and extracted by ultrasonic with 20 ml of 80% methanol solution for 45 min. Then the resultant mixture was adjusted to the original weight and aliquots of the supernatant were filtered through 0.45μm membrane before HPLC injection. The contents of verbenalin and hastatoside were determined by HPLC. The separation was carried out on a C18 column with the mixture of acetonitrile-water (15:85) as a mobile phase at the flow rate of 1.0 mL/min and detection wavelength was set at 238 nm. For simultaneous determination of luteolin, apigenin and kaempferol, the separation was performed by a C18 column using using gradient acetonitrile-0.1% formic acid as a mobile phase at 350 nm.Results: For HPLC, there were good linear relationships for verbenalin and hastatoside within the range of 17.2～155.2μg/mL and 25.5～229.9μg/mL. The average recoveries were 98.89% (RSD=1.1%), 99.00% (RSD=0.4%). For the luteolin, apigenin and kaempferol, the linear regressions were acquired with r2>0.995, respectively. The precision was evaluated and the relative standard deviation (RSD) values were reported within 2.0 %. The average recovery studies for the quantified compounds were observed as 98.14%, 100.3%, 97.12%, with RSD values less than 2.0 %.Conclution: These two methods were simple, accurate and reproducible. They can set the basis for reasonable application and quality control for Verbena Oficinalis L.Part three LC-MS/MS determination and pharmacokinetic study of five flavone components in rat plasma after oral administration of Verbena Oficinalis L. extractObjective: A selective and sensitive HPLC-MS/MS method was first developed and validated for determination of luteolin, kaempferol, apigenin, quercetol, and isorhamnetin in rat plasma. Blood samples were collected via the fossa orbitalis vein at time intervals after oral administration and the concentrations of the five ingredients in plasma were analyzed by HPLC-MS/MS after the plasma protein had been precipitated.Methods: Blood samples of about 0.3 mL were collected into heparinized centrifuge tubes from the fossa orbitalis vein at 5, 15, 30, 60, 90, 120, 150, 180, 240, 360, 480, 720 min in 6 healthy rats after single oral administration of Verbena Oficinalis L extract (10 mL/kg). SMZ was used as an internal standard (I.S.), and plasma samples were pretreated by a single-step protein precipitation with MeOH-Hydrochloric acid (25%) (4:1, v/v). Chromatographic separation was achieved on a Waters SunFireTM C18 column (150 mm×4.6 mm, 5μm) at 25°C with a mobile phase consisting of acetonitrile and 0.1% aqueous formic acid at a flow rate of 0.8 ml/min. The five flavone components were satisfactorily separated with a run time of 5 min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) under negative ionization mode with an electrospray ionization (ESI) source. The turbo spray temperature was set at 650℃with a ionspray voltage -5000 V. Nebulizer gas (gas 1) 60 psi, heater gas (gas 2) 65 psi and curtain gas (CUR) 25 psi. Nitrogen was used in all cases. The MS/MS transitions used for monitoring were at (m/z) 285.0/133.0 for luteolin, 285.0/92.9 for kaempferol, 269.0/117.0 for apigenin, 301.1/151.0 for quercetol, 315.1/303.3 for isorhamnetin, and 252.0/155.9 for IS.Results: Calibration curves of luteolin, kaempferol, apigenin, quercetol, and isorhamnetin were generated over the range 5-2500, 5-2500, 20-10000, 25-12500 and 20-10000 ng/mL, respectively. The lower limits of quantitation (LLOQ) of these analytes were less than 25 ng/mL. The mean extraction recoveries for all compounds were between 81.5%-108.1%. The intra- and inter-day precisions (RSD) for the analysis of five analytes were between 1.7% and 10.8% with accuracies (RE) between -8.3% and 6.9%. The matrix effect were between 87.80% and 107.6%. No matrix effect was observed in this method. Luteolin, kaempferol, apigenin, quercetol, and isorhamnetin were stable during all storage, pretreatment and analytical periods. The main pharmacokinetic parameters of luteolin, kaempferol, apigenin, quercetol, and isorhamnetin were as follows: T1/2 were 4.02±0.81,7.65±0.71, 3.30±0.83, 4.55±0.49 and 5.56±1.32 h, AUC0?t were 6377.71±302.49, 3624.10±248.08, 25875.79±487.54, 13442.09±787.12 and 2714.95±96.84 ngh/mL, AUC0?∞were 10674.84±344.42, 5140.93±852.06, 28767.20±206.33, 15806.8±265.17 and 3254.92±332.65 ngh/mL, MRT0?∞were 9.67±3.88, 12.75±1.43, 5.95±0.57, 5.06±1.03 and 7.09±1.53 h.Conclusion: The validated method has been successfully applied to a pharmacokinetic study of luteolin, kaempferol, apigenin, quercetol, and isorhamnetin after oral administration of Verbena Oficinalis L. extract to rats.Part four Drug-protein binding determination of luteolin and apigeninObjective: To develop a high performance liquid chromatography (HPLC) to determine the protein binding rates of luteolin and apigenin in human plasma, rat plasma, bovine serum albumin (BSA), and to calculate the correlate parameters of verbenalin and hastatoside to different genera plasma proteins.Methods: The binding rates of luteolin and apigenin with different genera plasma proteins were determined by equilibrium dialysis method. The concentrations of luteolin and apigenin were assayed by HPLC.Results: The binding rates of luteolin with rat, human plasma and BSA were 99.1±13.7%, 99.2±12.4%, 99.2±6.1%, 99.3±4.4%, 99.4±8.3%, 99.5±5.6%, 97.7±10.2%, 98.1±9.8%, 89.6±5.0%, respectively. The binding rates of apigenin with rat, human plasma and BSA were 99.4±3.8%, 99.6±5.6%, 99.5±4.5%, 96.3±7.2%, 97.9±10.5%, 97.8±9.7%, 98.7±8.6%, 99.6±9.4%, 99.1±8.0%.Conclusion: The equilibrium dialysis method was applied to study the the protein binding rates of luteolin and apigenin. The binding rates of luteolin and apigenin to human plasma protein, rat plasma protein and BSA were very high. They were a kind of high plasma protein binding rate drug and most of the drug molecules act on body with binding type. It would made the obvious change of free fraction with pharmacological action that the protein binding of luteolin and apigenin is not dependent on the doses. It is suggested that the above results may not ensure to have safety of luteolin and apigenin in clinic.