Studies On Antitussive Effect And The Pharmacodyamic Material Basis From 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 Chenghui Gui 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 exist extensively. Are there some parts else to show significant antitussive action?In this paper, the antitussive effect was studied systematically by giving animals the total extract and the partitions from Verbena Officinalis L. to find the pharmacodyamic material basis. The main components was isolated and purified to study their contents in Verbena Officinalis L. and the absorption, distribution of verbenalin and hastatoside in vivo were explored.Part one Experimental studies on antitussive effects of the extracts from Verbena officinalis L.Objective: To compare and observe antitussive and anti-inflammatory effects of the total extract and its fractions from Verbena Officinalis L.; to clarify the pharmacodyamic material basis and the mechanism.Methods: The effect of the total extract and its five parts was evaluated on cough induced by ammonia solution in mice. The anti-inflammatory effect of the extracts from Verbena officinalis L. was studied on ear edema induced by xylene in mice. The actions were further proved by cough induced by citric acid in guinea pig and the secretion of phenolred of trachea in mice. The effects on isolated tracheal contraction of guinea pig caused by histamine, potassium chloride or acetylcholine were observed.Results: The total extract and its ethyl acetate and n-butyl alcohol parts could obviously decreased the times of cough in mice and prolonged latent period and they also had significant anti-inflammatory effect by inhibitting the mouse xylene-induced ear swelling. Three doses of the total extract could lessenned the times of cough in guinea pig and extended its cough latent period and cut down the secretion of phenolred of trachea of mice. They could also markedly relieve the contraction of isolated tracheal smooth muscle of guinea pig induced by histamine except acetylcholine and potassium chloride.Conclusion: The total extract and its ethyl acetate and n-butyl alcohol parts have marked antitussive and anti-inflammatory effect.Part two Studies on the chemical constituents of bioacive parts of Verbena officinalis L.Objective: To isolate and purify the compounds of bioacive parts in Verbena officinalis L. by the technologies of Silica gel column chromatography, Sephadex-LH-20 gel column, chromatography, preparative TLC, HPLC and recrystallization and to characterize the structures of the isolated pure compounds by using the spectroscopic methods including MS, 1H-NMR, 13C-NMR.Methods: The pulverized dried aerial parts of Verbena officinalis L. (10 Kg) were macerated for 7 days at room temperature with 95% alcohol for two times. The leachate was heated and activated carbon was added to absorb and then filtered and alcohol was evaporated in vacuum to yield the total crude extract of 520 g. The crude extract was suspended in saturated brine and extracted successively with petroleum ether, trichloromethane, ethyl acetate and n-butyl alcohol in order to obtain four fractions: the petroleum ether fraction 11 g, the trichloromethane fraction 39 g, the ethyl acetate fraction 52 g and the n-butyl alcohol fraction 118 g. The ethyl acetate fraction and n-butyl alcohol fraction were applied to pass silica gel column chromatography for preliminary fractionation in turn; each fraction was monitored with TLC and the similar fractions were combined. The subfractions was combined and subjected to Silica gel column chromatography, Sephadex-LH-20 gel column chromatography, preparative TLC and/or reversed phase preparative HPLC for further separation and purification to get pure compounds. The spectroscopic methods including NMR methods were used for the structural identification of these compounds.Results: 12 compounds were yielded by systemical separation of the aerial parts of Verbena officinalis L. The structures of 12 compounds were identified on the basis of chemical and spectral analysis, including 6 flavones, 4 iridoid glycosides, a phenylpropanoid glycoside and a phytostero.Conclution: The result indicated that the solvents and methods of extraction and isolation used in this experiment were practicable. Silica gel column chromatography, Sephadex-LH-20 gel column chromatography, preparative TLC and HPLC were employed to isolate and purify the components of the aerial parts of Verbena officinalis L., and spectroscopic methods were used to establish the structures of the compounds. 12 compounds were obtained and identified with the aid of spectroscopic methods. They were kaempferol (1), apigenin (2), 4'-hydroxywogonin (3), quercitrin (4), luteoiin (5), Isorhamnetin (6), verbascoside (7), verbenalin (8), hastatoside (9), aucubin(10), gentiopicroside (11),β-sitosterol (12).Part three Simultaneous determination of main glycosides in Verbena officinalis L.Objective: To develop a high performance liquid chromatography coupled with variable wavelength detector (VWD) method for simultaneous determination of verbenalin and hastatoside in Verbena officinalis L.; to establish a high performance liquid chromatography coupled with electrospray tandem mass spectrometry for simultaneous determination of 5 glycosides (aucubin, hastatoside, verbenalin, gentiopicroside and verbascoside) 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. For HPLC-VWD, The contents of verbenalin and hastatoside were determined by HPLC. The separation was carried out on a C18 column with a 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 HPLC-MS-MS, the separation was performed by a C18 column using gradient acetonitrile-0.1 % formic acid as a mobile phase. In this study, the mass spectral conditions were optimized in both positive- and negative-ion modes, and the positive-ion mode was found to be more sensitive for aucubin, hastatoside, verbenalin and gentiopicroside and so negative-ion mode for verbascoside.Results: For HPLC-VWD, 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 HPLC-MS-MS, 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 101.2%, 98.63%, 99.45%, 101.6%, 100.3% 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 four Studies on pharmacokinetics and tissue distribution of verbenalin and hastatoside in ratsObjective: To develop a HPLC method for determination of verbenalin and hastatoside in rat plasma for studying the pharmacokinetics of verbenalin and hastatoside in rats after a single oral administration of verbenalin, hastatoside and the extract from Verbena Oficinalis L.; To develop and validate a HPLC method for determination of verbenalin and hastatoside in rat tissues for exploring tissue distribution of verbenalin and hastatoside in rats.Methods: After rats were orally administrated verbenalin, hastatoside and the extract from Verbena Oficinalis L., blood samples were obtained from fossa orbitalis vein according to the specific schedule: 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 and 360 min and collected in heparinized centrifuge tube, respectively. Sample was pretreated by protein precipitation with methanol using paeoniflorin as internal standar (IS) and the supernatant was collected and evaporated to dryness at 40℃under a gentle stream of nitrogen. The residue was then reconstituted with 100μL water, and centrifuged at 12000×g for 10 min, and an aliquot (20μL) of the supernatant was injected into the HPLC system. The RP-C18 column (250 mm×4.6mm,5μm) was used as the stationary phase with the mobile phase consisting of acetonitrile–water (15:85, v/v) at a flow rate of 1.0mL·min-1. Chromatograms were monitored at 238 nm for verbenalin and hastatoside and the temperature of column was kept at 35℃. The flow rate was 1.0mL·min-1. Rats were randomly assigned to six groups. The method was applied to study tissues distribution of verbenalin and hastatoside in rats after a single administration of verbenalin, hastatoside and the extract from Verbena Oficinalis L. at a dose of 40 mg·kg-1 for verbenalin and 52 mg·kg-1 for hastatoside. After oral administration of verbenalin, hastatoside and the extract from Verbena Oficinalis L., heart, liver, lung, spleen, kidney, brain, stomach and small intestine samples were obtained at 60, 180, 300 min, respectively. Tissue samples were weighed rapidly and put into normal saline solution to remove the blood or content, blotted on filter paper, and then were weighed for wet weight and homogenized in saline solution (500 mg·mL-1). Preparation of tissues samples and HPLC analysis conditon were same with the plasma samples.Results: The calibration curve of verbenalin and hastatoside in plasma were linear over the range of 31.2~625 and 20.3~812.5 ng·mL-1 and the RSD values of intra-day and inter-day were less than 15%. The study of stability demonstrated samples were stable. After a single oral administration of verbenalin, hastatoside and the extract from Verbena Oficinalis L. at the dose of 40 and 52 mg·kg-1 to rats, The 1.41-fold (38591.6 ng/ml/min versus 27177.5 ng/ml/min) enhancements of AUC0–t were observed from verbenalin compared with Verbena oficinalis L. extract at a dosage of 40 mg/kg verbenalin and 1.35-fold (51266.8 ng/ml/min versus 37885.7 ng/ml/min) enhancements of AUC0–t were observed from hastatoside compared with Verbena oficinalis L. extract at a dosage of 52 mg/kg hastatoside. The results indicated that verbenalin and hastatoside underwent a rapid and wide distribution in the tissues/organs throughout the whole body within the time course examined. Following 60 min of administration of verbenalin, hastatoside or Verbena oficinalis L. extract, all tissues analyzed contained a significant amount of verbenalin and hastatoside. Verbenalin and hastatoside showed substantial disposition in stomach, liver, heart, small intestine and spleen. The distribution of verbenalin and hastatoside in brain showed that it had the ability to cross the blood-brain barrier after oral administration.Conclusion: The bioavailabilities of verbenalin and hastatoside were higher than those of verbenalin and hastatoside in Verbena oficinalis L. extract after they were given to rats. Stomach, liver, heart, small intestine and spleen were the main distribution tissues of verbenalin and hastatoside in rats, and verbenalin and hastatoside were found to cross the blood brain barrier. It was also found there was no accumulation of verbenalin and hastatoside in rat tissues.Part five Drug-protein binding determination of verbenalin and hastatosideObjective: To develop a high performance liquid chromatography (HPLC) to determine the protein binding rates of verbenalin and hastatoside 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 verbenalin and hastatoside with different genera plasma proteins were determined by equilibrium dialysis method. The concentrations of verbenalin and hastatoside were assayed by HPLC.Results: The binding rates of verbenalin with rat, human plasma and BSA were 15.42±12.8%, 17.27±12.6%, 12.69±4.1%, 30.53±3.6%, 13.59±8.8%, 24.09±7.3%, 20.45±7.2%, 17.24±10.8%, 16.89±6.0%, respectively. The binding rates of hastatoside with rat, human plasma and BSA were 20.91±3.9%, 25.60±5.4%, 19.52±4.7%, 24.30±7.6%, 21.76±6.5%, 23.12±5.7%, 26.19±5.6%, 27.70±9.1%, 25.83±7.0%.Conclusion: The equilibrium dialysis method was applied to study the the protein binding rates of verbenalin and hastatoside. The binding rates of verbenalin and hastatoside to human plasma protein, rat plasma protein and BSA were very low. They were a kind of low plasma protein binding rate drug and most of the drug molecules act on body with free type. It would not made the obvious change of free fraction with pharmacological action that the protein binding of verbenalin and hastatoside is not dependent on the doses.It is suggested that the above results may ensure to have safety of verbenalin and hastatoside in clinic.Part six Studies on Absorption Kinetics of verbenalin and Hastatoside in Intestines in RatsObjective: To develop a high-performance liquid chromatography coupled with diode array detection (HPLC/DAD) method for simultaneous determination of verbenalin or hastatoside and phenolsulfonphthalein in the circulation solution and to investigate the absorption kinetics of verbenalin and hastatoside at different intestine segments of rats and the influence of the drug solution concentration on the absorption kinetics.Methods: The intestine in rats was cannulated for in situ recirculation. The concentrations of verbenalin, hastatoside or phenolsulfonphthalein in the flux were measured by the reversed phase HPLC. The chromatographic procedure was carried out with Diamonsil 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 at the temperature of column of 30?C. The detection wavelength was set at 238 nm for verbenalin or hastatoside and 430 nm for phenolsulfonphthalein and the flow rate was 1.0 mL/min.Results: When the concentration was raised from 0.05 to 0.20 mg?mL-1, the uptake of verbenalin and hastatoside was increased linearly. Concentration had no effect on the permeability coefficient. The permeability coefficients of verbenalin at duodenum, jejunum, ileum and colon were (0.0536±0.0062), (0.0504±0.0051), (0.0523±0.0037), (0.0492±0.0023) h-1 and those of hastatoside were (0.0405±0.0039), (0.0365±0.0032), (0.0379±0.0045) and (0.0349±0.003) h-1, respectively.Conclusion: It is the first time to use sensitive, accurate, and simple HPLC/DAD method to determine verbenalin or hastatoside and phenolsulfonphthalein in the circulation solution simultaneously. The absorption of verbenalin and hastatoside in rat's intestine is a first-order process with the passive diffusion mechanism. Verbenalin and hastatoside can be absorbed in whole intestinal segments. So verbenalin and hastatoside sustained-released formulations can be prepared.