马鞭草镇咳作用及药效物质基础研究
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摘要
马鞭草为马鞭草科(Verbenaceae)植物马鞭草Verbena officinalis L.的干燥地上部分,具有活血散瘀,截疟,解毒,利水消肿的功效。《Herbal Drugs and Phytopharmaceuticals》记载,在民间该药常常被用作利尿药、祛痰药和抗风湿药,在西班牙的Navarra地区,该药广泛用于抗炎。桂承会等于1985年报道马鞭草水煎剂有一定镇咳作用,并证明马鞭草苷的镇咳作用与水煎剂基本一致。一般而言,中药发挥药效作用的物质基础多为一类或几类物质成分,马鞭草苷为马鞭草所含成分之一,与其同类的其他成分也有很多,是否发挥镇咳作用的为包括马鞭草苷在内的糖苷类物质呢?
本文通过研究马鞭草提取液及分取液的镇咳活性,寻找其药效物质基础,在此基础上,分离确定马鞭草有效部位的化学成分,并对主要活性物质的含量、在动物体内的吸收、分布等情况进行探讨,为马鞭草的开发利用奠定基础。
第一部分马鞭草镇咳作用的研究
目的:研究马鞭草镇咳作用及有效部位,明确马鞭草镇咳作用的药效物质基础。
方法:观察马鞭草总提物及分取部位对小鼠因氨水所致咳嗽以及对二甲苯致小鼠耳肿胀的影响;利用豚鼠因枸橼酸所致咳嗽试验对不同剂量的马鞭草提取物的镇咳和咳嗽潜伏期进行考察;利用小鼠呼吸道酚红排痰量试验研究马鞭草提取物的祛痰作用;探讨马鞭草提取物对磷酸组胺、氯化乙酰胆碱、氯化钾所致豚鼠离体气管痉挛的影响,分析其作用机制。
结果:与对照组比较,马鞭草水提物、马鞭草醇提物、乙酸乙酯分取液、正丁醇分取液能明显减少小鼠咳嗽次数,显示出一定的镇咳效果,显著延长浓氨水诱发小鼠咳嗽潜伏期;与对照组比较,马鞭草水提物、马鞭草醇提物、石油醚分取液、氯仿分取液、乙酸乙酯分取液、正丁醇分取液能明显抑制小鼠耳朵的肿胀度,显示出一定的抗炎效果;与对照组相比,马鞭草提取物小、中、大剂量明显减少豚鼠咳嗽次数,显示出一定的镇咳效果并能显著延长枸橼酸诱发豚鼠咳嗽潜伏期;与对照组相比,马鞭草醇提物能明显增加酚红气道排泄量,马鞭草醇提物显示出一定的祛痰作用;不同剂量的马鞭草醇提物对磷酸组胺引起的离体气管平滑肌痉挛均有一定程度的抑制作用,而对氯化乙酰胆碱和氯化钾引发气管平滑肌痉挛没有作用。
结论:药效学研究结果表明,马鞭草能够抑制浓氨水所致小鼠咳嗽、枸橼酸所致豚鼠的咳嗽反应,并有明显的祛痰、对抗二甲苯所致小鼠耳廓肿胀的作用,能够显著地对抗磷酸组胺所致的豚鼠离体气管平滑肌痉挛。马鞭草的正丁醇部位和乙酸乙酯部位为发挥药效作用的主要物质基础。
第二部分马鞭草镇咳有效部位化学成分的研究
目的:本研究以马鞭草正丁醇和乙酸乙酯部分为研究对象,利用硅胶柱色谱、葡聚糖凝胶色谱、制备薄层色谱、高效液相制备色谱以及重结晶等分离纯化技术对其进行分离研究,寻找含有的化学成分,并应用MS、1H-NMR、13C-NMR现代化手段完成单体化合物分子的结构鉴定,为进一步开发利用提供依据。
方法:干燥的马鞭草(10 kg)适当粉碎后,用95%乙醇冷浸二次,浸出液加热,加入活性炭趁热抽滤,合并滤液减压浓缩至粘膏状(520 g),将其悬浮于饱和NaCl水溶液中,分别用石油醚、氯仿、乙酸乙酯和正丁醇萃取,继而得到石油醚部位(11 g)、氯仿部位(39 g)、乙酸乙酯部位(52 g)和正丁醇部位(118 g)。本研究先后对乙酸乙酯部位和正丁醇部位采用硅胶柱色谱进行初步分离,经薄层色谱检识后,进一步用硅胶柱色谱、葡聚糖凝胶色谱、制备薄层色谱、高效液相制备色谱反复分离纯化得到单体化合物。运用MS、1H-NMR、13C-NMR波谱学方法进行结构鉴定。
结果:通过系统提取分离马鞭草,从中得到12个单体化合物,包括6个黄酮化合物,4个环烯醚萜苷类化合物,1个苯丙素糖苷,1个甾醇。
结论:本文对马鞭草的化学成分进行了研究,所选用的提取方法及提取溶剂适当,采用了硅胶柱色谱、葡聚糖凝胶色谱、制备薄层色谱、制备液相色谱等分离手段,共分离得到12个化合物,并且运用多种现代波谱手段及其相关知识进行了结构鉴定,包括6个黄酮化合物,4个环烯醚萜苷类化合物,1个苯丙素糖苷,1个甾醇:山奈素,芹菜素,异鼠李素,4'-羟基汉黄芩素,槲皮苷,木犀草素,马鞭草苷,戟叶马鞭草苷,桃叶珊瑚苷,龙胆苦苷,毛蕊花糖苷,β-谷甾醇。
第三部分马鞭草主要糖苷含量测定的研究
目的:建立HPLC/VWD法同时测定马鞭草中马鞭草苷和戟叶马鞭草苷以及利用HPLC/MS/MS法同时测定马鞭草中桃叶珊瑚苷、马鞭草苷、戟叶马鞭草苷、龙胆苦苷、毛蕊花糖苷的含量的方法,以控制该药材的质量。
方法:取马鞭草药材粉末约0.5 g,精密称定,加入80%甲醇20ml,密塞,称定重量,超声处理45 min,放冷,称重,用80%甲醇补充减失的重量,摇匀,0.45μm微孔滤膜滤过,取续滤液,进样测定。HPLC/VWD法采用Diamonsil C18色谱柱(250 mm×4.6 mm , 5μm) ,乙腈-水(15∶85)为流动相,检测波长为238 nm。HPLC/MS/MS法采用色谱柱Waters SunfireTM C1(8150 mm×4.6 mm, 5μm),以乙腈-0.1%甲酸溶液(25:75→40:60)(0→6 min)为流动相进行梯度洗脱,流速0.8 ml/min,进样量5μl。
结果:对于HPLC/VWD法马鞭草苷、戟叶马鞭草苷在17.2～155.2μg/mL和25.5～229.9μg/mL的浓度范围内与峰面积均呈良好的线性关系;平均回收率分别为98.89%(RSD=1.1%)、99.00%(RSD=0.4%)。对于HPLC/MS/MS法,桃叶珊瑚苷、马鞭草苷、戟叶马鞭草苷、龙胆苦苷、毛蕊花糖苷五种组分的线性关系均良好(r2>0.995),平均回收率分别为:101.2%、98.63%、99.45%、101.6%、100.3%,精密度和重复性均良好。
结论:本部分利用HPLC/VWD法建立了同时测定马鞭草中马鞭草苷和戟叶马鞭草苷含量的方法,并利用HPLC/MS/MS建立了马鞭草中马鞭草苷、戟叶马鞭草苷、桃叶珊瑚苷、龙胆苦苷、毛蕊花糖苷同时测定的方法,二者均有较高的专属性、灵敏度,具有较好的精密度和准确度,能够满足马鞭草药材质量控制的要求。
第四部分马鞭草苷和戟叶马鞭草苷药代动力学与组织分布的研究
目的:建立高效液相色谱法测定大鼠血浆中马鞭草苷和戟叶马鞭草苷浓度的方法,并研究马鞭草苷和戟叶马鞭草苷单体以及马鞭草提取物单次给药后在大鼠体内的药代动力学过程;研究马鞭草苷和戟叶马鞭草苷在大鼠体内的组织分布。
方法:大鼠以灌胃给予马鞭草苷和戟叶马鞭草苷单体以及马鞭草提取物,分别于给药后30、60、90、120、150、180、210、240、270、300、330、360 min内眦动脉取血,置于肝素化塑料离心管中,样品预处理采用甲醇沉淀蛋白后氮气吹干,水复溶,利用HPLC内标法(芍药苷),色谱柱为反相C18柱(250mm×4.6mm,5μm),流动相为乙腈-水(15:85),流速为1.0mL·min-1,检测波长238nm;取大鼠随机分为6组,灌胃给予马鞭草苷和戟叶马鞭草苷单体以及马鞭草提取物后,于60、180、300 min分别放血处死,取心、肝、肺、脾、肾、脑、胃和肠,经生理盐水冲洗,去除少量的淤血或组织内容物,并用滤纸吸去水分,称重后于匀浆器中制成500mg·mL-1的生理盐水匀浆,采用甲醇沉淀蛋白后氮气吹干水复溶的方法,利用HPLC法(同上方法)测定组织中的马鞭草苷和戟叶马鞭草苷含量。
结果:血浆中马鞭草苷和戟叶马鞭草苷的线性范围31.2～625、20.3～812.5 ng·mL-1,定量下限为31.2、20.3 ng·mL-1,马鞭草苷31.2、125、625 ng·mL-1三种浓度的日内、日间精密度(RSD)分别为5.64%~7.18%和4.89%~8.99%,准确度(RE)为-1.0%~9.5%,戟叶马鞭草苷20.3、162、812.5 ng·mL-1三种浓度的日内、日间精密度(RSD)分别为0.53%~5.62%和0.59%~4.98%,准确度(RE)为-0.3%~9.5%,稳定性研究结果表明样品稳定。大鼠单次按马鞭草苷40 mg·kg-1、戟叶马鞭草苷52 mg·kg-1分别灌胃马鞭草苷、戟叶马鞭草苷单体和马鞭草提取物后,马鞭草苷、戟叶马鞭草苷的AUC及Cmax明显高于马鞭草提取物。对于马鞭草苷,给予单体后的AUC值是马鞭草提取物的1.41倍(38591.6 ng/ml/min对27177.5 ng/ml/min),对于戟叶马鞭草苷,则为1.35倍(51266.8 ng/ml/min对37885.7 ng/ml/min)。大鼠灌胃按马鞭草苷40 mg·kg-1和按戟叶马鞭草苷52 mg·kg-1给予马鞭草苷、戟叶马鞭草苷单体和马鞭草提取物后,马鞭草苷和戟叶马鞭草苷在大鼠体内经历了一个广泛而快速的分布过程,即在第一个检测时间60 min就能在各组织中发现马鞭草苷和戟叶马鞭草苷,但是它们主要分布在胃、肝、心、小肠及脾。在脑中的分布显示出马鞭草苷和戟叶马鞭草苷能透过血脑屏障。
结论:灌胃后马鞭草苷和戟叶马鞭草苷单体生物利用度明显高于马鞭草提取物中的马鞭草苷和戟叶马鞭草苷;马鞭草苷和戟叶马鞭草苷主要分布在胃、肝、心、小肠及脾。在脑中的分布表示马鞭草苷和戟叶马鞭草苷能透过血脑屏障。在所研究的组织中,未见明显的特殊蓄积现象。
第五部分马鞭草苷和戟叶马鞭草苷血浆蛋白结合率的测定
目的:建立马鞭草苷和戟叶马鞭草苷在大鼠血浆、人血浆和牛血清白蛋白中蛋白结合率的测定方法,并计算不同种属血浆蛋白的相关参数。
方法:采用平衡透析法测定血浆蛋白结合率,用高效液相色谱法测定血浆中药物总浓度及游离的药物浓度。
结果:马鞭草苷的血浆蛋白结合率分别为:大鼠血浆:15.42±12.8%、17.27±12.6%、12.69±4.1%;人血浆:30.53±3.6%、13.59±8.8%、24.09±7.3%;牛血清白蛋白:20.45±7.2%、17.24±10.8%、16.89±6.0%。戟叶马鞭草苷的血浆蛋白结合率分别为:大鼠血浆:20.91±3.9%、25.60±5.4%、19.52±4.7%;人血浆:24.30±7.6%、21.76±6.5%、23.12±5.7%;牛血清白蛋白:26.19±5.6%、27.70±9.1%、25.83±7.0%。
结论:本文应用平衡透析法研究了马鞭草苷和戟叶马鞭草苷与人血浆蛋白、牛血清白蛋白和大鼠血浆蛋白的结合情况,结果表明马鞭草苷和戟叶马鞭草苷的血浆蛋白结合率较低,属低血浆蛋白结合率药物,大部分药物分子以游离形式发挥药效,并且不具有浓度依赖性,不易引起具有药理作用的游离型血药浓度发生明显变化,说明马鞭草苷和戟叶马鞭草苷临床用药有较好的安全性。
第六部分马鞭草苷和戟叶马鞭草苷大鼠在体肠吸收动力学的研究
目的:建立同时测定肠循环液中马鞭草苷或戟叶马鞭草苷与酚红浓度的HPLC/DAD法,探讨马鞭草苷和戟叶马鞭草苷在大鼠各肠段的吸收动力学特征及不同药物浓度对其的影响。
方法:采用大鼠在体肠吸收实验方法,以HPLC/DAD法测定肠循环液中药物的含量,色谱条件为:Diamonsil C18色谱柱(250 mm×4.6 mm, 5μm);柱温30℃;流动相为乙腈:0.05%磷酸溶液,梯度洗脱,流速1.0 mL/min;检测波长238 nm(马鞭草苷或戟叶马鞭草苷)和430 nm(酚红);进样量20μL。
结果:在0.05～0.20 mg/mL浓度内马鞭草苷或戟叶马鞭草苷的吸收量与浓度成线性关系,Ka值基本保持不变;马鞭草苷和戟叶马鞭草苷各肠段的吸收速率无显著性差异,马鞭草苷在十二指肠、空肠、回肠、结肠的Ka值分别为(0.0536±0.0062),(0.0504±0.0051),(0.0523±0.0037),(0.0492±0.0023)h-1;戟叶马鞭草苷在十二指肠、空肠、回肠和结肠的Ka值分别为(0.0405±0.0039),(0.0365±0.0032),(0.0379±0.0045)和(0.0349±0.0037)h-1。
结论:本文首次建立了HPLC/DAD法同时测定肠循环液中马鞭草苷或戟叶马鞭草苷及酚红的浓度的方法,该法操作简便,结果准确,灵敏度高。研究结果表明,马鞭草苷和戟叶马鞭草苷在肠道的吸收呈现一级动力学过程,且吸收机制为被动扩散;马鞭草苷和戟叶马鞭草苷在整个肠道均有吸收,可以将马鞭草苷或戟叶马鞭草苷研制成缓释制剂。
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 rats
Objective: 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 hastatoside
Objective: 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 Rats
Objective: 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.
本文通过研究马鞭草提取液及分取液的镇咳活性,寻找其药效物质基础,在此基础上,分离确定马鞭草有效部位的化学成分,并对主要活性物质的含量、在动物体内的吸收、分布等情况进行探讨,为马鞭草的开发利用奠定基础。
第一部分马鞭草镇咳作用的研究
目的:研究马鞭草镇咳作用及有效部位,明确马鞭草镇咳作用的药效物质基础。
方法:观察马鞭草总提物及分取部位对小鼠因氨水所致咳嗽以及对二甲苯致小鼠耳肿胀的影响;利用豚鼠因枸橼酸所致咳嗽试验对不同剂量的马鞭草提取物的镇咳和咳嗽潜伏期进行考察;利用小鼠呼吸道酚红排痰量试验研究马鞭草提取物的祛痰作用;探讨马鞭草提取物对磷酸组胺、氯化乙酰胆碱、氯化钾所致豚鼠离体气管痉挛的影响,分析其作用机制。
结果:与对照组比较,马鞭草水提物、马鞭草醇提物、乙酸乙酯分取液、正丁醇分取液能明显减少小鼠咳嗽次数,显示出一定的镇咳效果,显著延长浓氨水诱发小鼠咳嗽潜伏期;与对照组比较,马鞭草水提物、马鞭草醇提物、石油醚分取液、氯仿分取液、乙酸乙酯分取液、正丁醇分取液能明显抑制小鼠耳朵的肿胀度,显示出一定的抗炎效果;与对照组相比,马鞭草提取物小、中、大剂量明显减少豚鼠咳嗽次数,显示出一定的镇咳效果并能显著延长枸橼酸诱发豚鼠咳嗽潜伏期;与对照组相比,马鞭草醇提物能明显增加酚红气道排泄量,马鞭草醇提物显示出一定的祛痰作用;不同剂量的马鞭草醇提物对磷酸组胺引起的离体气管平滑肌痉挛均有一定程度的抑制作用,而对氯化乙酰胆碱和氯化钾引发气管平滑肌痉挛没有作用。
结论:药效学研究结果表明,马鞭草能够抑制浓氨水所致小鼠咳嗽、枸橼酸所致豚鼠的咳嗽反应,并有明显的祛痰、对抗二甲苯所致小鼠耳廓肿胀的作用,能够显著地对抗磷酸组胺所致的豚鼠离体气管平滑肌痉挛。马鞭草的正丁醇部位和乙酸乙酯部位为发挥药效作用的主要物质基础。
第二部分马鞭草镇咳有效部位化学成分的研究
目的:本研究以马鞭草正丁醇和乙酸乙酯部分为研究对象,利用硅胶柱色谱、葡聚糖凝胶色谱、制备薄层色谱、高效液相制备色谱以及重结晶等分离纯化技术对其进行分离研究,寻找含有的化学成分,并应用MS、1H-NMR、13C-NMR现代化手段完成单体化合物分子的结构鉴定,为进一步开发利用提供依据。
方法:干燥的马鞭草(10 kg)适当粉碎后,用95%乙醇冷浸二次,浸出液加热,加入活性炭趁热抽滤,合并滤液减压浓缩至粘膏状(520 g),将其悬浮于饱和NaCl水溶液中,分别用石油醚、氯仿、乙酸乙酯和正丁醇萃取,继而得到石油醚部位(11 g)、氯仿部位(39 g)、乙酸乙酯部位(52 g)和正丁醇部位(118 g)。本研究先后对乙酸乙酯部位和正丁醇部位采用硅胶柱色谱进行初步分离,经薄层色谱检识后,进一步用硅胶柱色谱、葡聚糖凝胶色谱、制备薄层色谱、高效液相制备色谱反复分离纯化得到单体化合物。运用MS、1H-NMR、13C-NMR波谱学方法进行结构鉴定。
结果:通过系统提取分离马鞭草,从中得到12个单体化合物,包括6个黄酮化合物,4个环烯醚萜苷类化合物,1个苯丙素糖苷,1个甾醇。
结论:本文对马鞭草的化学成分进行了研究,所选用的提取方法及提取溶剂适当,采用了硅胶柱色谱、葡聚糖凝胶色谱、制备薄层色谱、制备液相色谱等分离手段,共分离得到12个化合物,并且运用多种现代波谱手段及其相关知识进行了结构鉴定,包括6个黄酮化合物,4个环烯醚萜苷类化合物,1个苯丙素糖苷,1个甾醇:山奈素,芹菜素,异鼠李素,4'-羟基汉黄芩素,槲皮苷,木犀草素,马鞭草苷,戟叶马鞭草苷,桃叶珊瑚苷,龙胆苦苷,毛蕊花糖苷,β-谷甾醇。
第三部分马鞭草主要糖苷含量测定的研究
目的:建立HPLC/VWD法同时测定马鞭草中马鞭草苷和戟叶马鞭草苷以及利用HPLC/MS/MS法同时测定马鞭草中桃叶珊瑚苷、马鞭草苷、戟叶马鞭草苷、龙胆苦苷、毛蕊花糖苷的含量的方法,以控制该药材的质量。
方法:取马鞭草药材粉末约0.5 g,精密称定,加入80%甲醇20ml,密塞,称定重量,超声处理45 min,放冷,称重,用80%甲醇补充减失的重量,摇匀,0.45μm微孔滤膜滤过,取续滤液,进样测定。HPLC/VWD法采用Diamonsil C18色谱柱(250 mm×4.6 mm , 5μm) ,乙腈-水(15∶85)为流动相,检测波长为238 nm。HPLC/MS/MS法采用色谱柱Waters SunfireTM C1(8150 mm×4.6 mm, 5μm),以乙腈-0.1%甲酸溶液(25:75→40:60)(0→6 min)为流动相进行梯度洗脱,流速0.8 ml/min,进样量5μl。
结果:对于HPLC/VWD法马鞭草苷、戟叶马鞭草苷在17.2～155.2μg/mL和25.5～229.9μg/mL的浓度范围内与峰面积均呈良好的线性关系;平均回收率分别为98.89%(RSD=1.1%)、99.00%(RSD=0.4%)。对于HPLC/MS/MS法,桃叶珊瑚苷、马鞭草苷、戟叶马鞭草苷、龙胆苦苷、毛蕊花糖苷五种组分的线性关系均良好(r2>0.995),平均回收率分别为:101.2%、98.63%、99.45%、101.6%、100.3%,精密度和重复性均良好。
结论:本部分利用HPLC/VWD法建立了同时测定马鞭草中马鞭草苷和戟叶马鞭草苷含量的方法,并利用HPLC/MS/MS建立了马鞭草中马鞭草苷、戟叶马鞭草苷、桃叶珊瑚苷、龙胆苦苷、毛蕊花糖苷同时测定的方法,二者均有较高的专属性、灵敏度,具有较好的精密度和准确度,能够满足马鞭草药材质量控制的要求。
第四部分马鞭草苷和戟叶马鞭草苷药代动力学与组织分布的研究
目的:建立高效液相色谱法测定大鼠血浆中马鞭草苷和戟叶马鞭草苷浓度的方法,并研究马鞭草苷和戟叶马鞭草苷单体以及马鞭草提取物单次给药后在大鼠体内的药代动力学过程;研究马鞭草苷和戟叶马鞭草苷在大鼠体内的组织分布。
方法:大鼠以灌胃给予马鞭草苷和戟叶马鞭草苷单体以及马鞭草提取物,分别于给药后30、60、90、120、150、180、210、240、270、300、330、360 min内眦动脉取血,置于肝素化塑料离心管中,样品预处理采用甲醇沉淀蛋白后氮气吹干,水复溶,利用HPLC内标法(芍药苷),色谱柱为反相C18柱(250mm×4.6mm,5μm),流动相为乙腈-水(15:85),流速为1.0mL·min-1,检测波长238nm;取大鼠随机分为6组,灌胃给予马鞭草苷和戟叶马鞭草苷单体以及马鞭草提取物后,于60、180、300 min分别放血处死,取心、肝、肺、脾、肾、脑、胃和肠,经生理盐水冲洗,去除少量的淤血或组织内容物,并用滤纸吸去水分,称重后于匀浆器中制成500mg·mL-1的生理盐水匀浆,采用甲醇沉淀蛋白后氮气吹干水复溶的方法,利用HPLC法(同上方法)测定组织中的马鞭草苷和戟叶马鞭草苷含量。
结果:血浆中马鞭草苷和戟叶马鞭草苷的线性范围31.2～625、20.3～812.5 ng·mL-1,定量下限为31.2、20.3 ng·mL-1,马鞭草苷31.2、125、625 ng·mL-1三种浓度的日内、日间精密度(RSD)分别为5.64%~7.18%和4.89%~8.99%,准确度(RE)为-1.0%~9.5%,戟叶马鞭草苷20.3、162、812.5 ng·mL-1三种浓度的日内、日间精密度(RSD)分别为0.53%~5.62%和0.59%~4.98%,准确度(RE)为-0.3%~9.5%,稳定性研究结果表明样品稳定。大鼠单次按马鞭草苷40 mg·kg-1、戟叶马鞭草苷52 mg·kg-1分别灌胃马鞭草苷、戟叶马鞭草苷单体和马鞭草提取物后,马鞭草苷、戟叶马鞭草苷的AUC及Cmax明显高于马鞭草提取物。对于马鞭草苷,给予单体后的AUC值是马鞭草提取物的1.41倍(38591.6 ng/ml/min对27177.5 ng/ml/min),对于戟叶马鞭草苷,则为1.35倍(51266.8 ng/ml/min对37885.7 ng/ml/min)。大鼠灌胃按马鞭草苷40 mg·kg-1和按戟叶马鞭草苷52 mg·kg-1给予马鞭草苷、戟叶马鞭草苷单体和马鞭草提取物后,马鞭草苷和戟叶马鞭草苷在大鼠体内经历了一个广泛而快速的分布过程,即在第一个检测时间60 min就能在各组织中发现马鞭草苷和戟叶马鞭草苷,但是它们主要分布在胃、肝、心、小肠及脾。在脑中的分布显示出马鞭草苷和戟叶马鞭草苷能透过血脑屏障。
结论:灌胃后马鞭草苷和戟叶马鞭草苷单体生物利用度明显高于马鞭草提取物中的马鞭草苷和戟叶马鞭草苷;马鞭草苷和戟叶马鞭草苷主要分布在胃、肝、心、小肠及脾。在脑中的分布表示马鞭草苷和戟叶马鞭草苷能透过血脑屏障。在所研究的组织中,未见明显的特殊蓄积现象。
第五部分马鞭草苷和戟叶马鞭草苷血浆蛋白结合率的测定
目的:建立马鞭草苷和戟叶马鞭草苷在大鼠血浆、人血浆和牛血清白蛋白中蛋白结合率的测定方法,并计算不同种属血浆蛋白的相关参数。
方法:采用平衡透析法测定血浆蛋白结合率,用高效液相色谱法测定血浆中药物总浓度及游离的药物浓度。
结果:马鞭草苷的血浆蛋白结合率分别为:大鼠血浆:15.42±12.8%、17.27±12.6%、12.69±4.1%;人血浆:30.53±3.6%、13.59±8.8%、24.09±7.3%;牛血清白蛋白:20.45±7.2%、17.24±10.8%、16.89±6.0%。戟叶马鞭草苷的血浆蛋白结合率分别为:大鼠血浆:20.91±3.9%、25.60±5.4%、19.52±4.7%;人血浆:24.30±7.6%、21.76±6.5%、23.12±5.7%;牛血清白蛋白:26.19±5.6%、27.70±9.1%、25.83±7.0%。
结论:本文应用平衡透析法研究了马鞭草苷和戟叶马鞭草苷与人血浆蛋白、牛血清白蛋白和大鼠血浆蛋白的结合情况,结果表明马鞭草苷和戟叶马鞭草苷的血浆蛋白结合率较低,属低血浆蛋白结合率药物,大部分药物分子以游离形式发挥药效,并且不具有浓度依赖性,不易引起具有药理作用的游离型血药浓度发生明显变化,说明马鞭草苷和戟叶马鞭草苷临床用药有较好的安全性。
第六部分马鞭草苷和戟叶马鞭草苷大鼠在体肠吸收动力学的研究
目的:建立同时测定肠循环液中马鞭草苷或戟叶马鞭草苷与酚红浓度的HPLC/DAD法,探讨马鞭草苷和戟叶马鞭草苷在大鼠各肠段的吸收动力学特征及不同药物浓度对其的影响。
方法:采用大鼠在体肠吸收实验方法,以HPLC/DAD法测定肠循环液中药物的含量,色谱条件为:Diamonsil C18色谱柱(250 mm×4.6 mm, 5μm);柱温30℃;流动相为乙腈:0.05%磷酸溶液,梯度洗脱,流速1.0 mL/min;检测波长238 nm(马鞭草苷或戟叶马鞭草苷)和430 nm(酚红);进样量20μL。
结果:在0.05～0.20 mg/mL浓度内马鞭草苷或戟叶马鞭草苷的吸收量与浓度成线性关系,Ka值基本保持不变;马鞭草苷和戟叶马鞭草苷各肠段的吸收速率无显著性差异,马鞭草苷在十二指肠、空肠、回肠、结肠的Ka值分别为(0.0536±0.0062),(0.0504±0.0051),(0.0523±0.0037),(0.0492±0.0023)h-1;戟叶马鞭草苷在十二指肠、空肠、回肠和结肠的Ka值分别为(0.0405±0.0039),(0.0365±0.0032),(0.0379±0.0045)和(0.0349±0.0037)h-1。
结论:本文首次建立了HPLC/DAD法同时测定肠循环液中马鞭草苷或戟叶马鞭草苷及酚红的浓度的方法,该法操作简便,结果准确,灵敏度高。研究结果表明,马鞭草苷和戟叶马鞭草苷在肠道的吸收呈现一级动力学过程,且吸收机制为被动扩散;马鞭草苷和戟叶马鞭草苷在整个肠道均有吸收,可以将马鞭草苷或戟叶马鞭草苷研制成缓释制剂。
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 rats
Objective: 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 hastatoside
Objective: 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 Rats
Objective: 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.
引文
1赵勤萍,张荣.浅谈中药有效部位研究对中药产业现代化的影响.山西中医,2006,22(3):48
2桂承会,唐人九.马鞭草镇咳有效成分的研究.中药通报, 1985,10(10): 35
3陈奇.中药药理研究方法学(第1版) .北京:人民卫生出版社,1994
4徐淑云.药理实验方法学(第2版) .北京:人民卫生出版社,2002,30
5冯冰虹,苏浩冲,杨俊杰.桑白皮丙酮提取物对呼吸系统的药理作用.广东药学院学报. 2005.21(1):47～49
6王立为,刘新民,余世春,等.枇杷叶抗炎和止咳作用研究.中草药. 2004,35(2): 174～176
7金正男,朴世浩,金景姬,等.引流熊胆的抗炎免疫作用.中国药理学通报. 1994,10(2): 143～146
8沈雅琴.苦刺总生物碱的抗炎作用.中国药理学通报. 1990,6(1): 10
9谢强敏,卞如廉,杨秋火,等.艾叶油的呼吸系统药理研究I,支气管扩张、镇咳和祛痰作用.中国现代应用药学杂志. 1999,16 (4):16-19
10 Enger H. and szelenyi l,Tracheal Phenolred secretion,a new methods for secreening mucosecretolytic compounds,of Pharmacological methods,Elsevier science Publishing Co,Newyork,1984,151
11周小江,曾南,贾敏如肺经草止咳化痰有效部位的初步筛选.中国民族民间医药杂志.2005,72: 46-49
12陈奇.中药药理研究方法学(第一版).人民卫生出版社,2000,7:651-652
13滕赞,李智.滨篙内醋对气管平滑肌的舒张作用及其细胞内钙的影响,2005,5: 13
14赵明沂,李智,丛华,等.滨篙内酷对豚鼠离体气管平滑肌张力的影响.中国药理学会通讯,2000,17(2):64
15潘泽等.国外疾学-内科学分册. 1999,26(3):11
1唐人九.人字草黄酮类化学成分研究.华西药学杂志,1996,11(1) :51
2喻蓉,许庆,李伯刚,等.搭棚藤的化学成分研究.天然产物研究与开发,2003,15(5) :4051
3许凤鸣,王兆全,李有文.半枝莲化学成分的研究.中国现代应用药学,1997,14(6) :81
4田菁,赵毅民,栾新慧.马鞭草化学成份的研究(Ⅱ).天然产物研究与开发, 2007,19:247-249
5陈改敏,张建业,张向沛,刘宏民.马鞭草黄酮类化学成分的研究.中药材,2006,29(7): 677-679
6于德泉,杨竣山.分析化学手册第二版.第七分册.核磁共振波谱分析.北京:化学工业出版社,1999,818,820
7蒋受军,朱彬,魏锋,等.小野芝麻化学成分的研究(I).中国中药杂志,2002, 27(9): 671-673
8 A. Bianco, M. Guiso, P. Passacantilli. Iridoid and phenylpropanoid glycosides from new sources. J. Nat. Prod., 1984, 47(5): 901-2
9 M. F. Lahloub, O. M. Salama, E. S. Mansour. Phenyl propanoid and iridoid glycosides from Verbena officinalis L. herb growing in Egypt. Bull. Fac. Pharm., 1990, 28(2): 75-7
10桂承会,唐人九.马鞭草镇咳成分的研究.中药通报,1985,10(10) :351
11 Dirk Teborg, Peter Junio. Iridoid glucosides from Penstemon nittdus. Planta Med, 1991, 57 :1841
12 Dirk Teborg, Peter Junior1 Iridoid glucosides from Penstemon nittdus. Planta Med , 1991, 57 :1841
1张涛,谢虹,李万.马鞭草提取物抗大鼠早孕作用的研究.中国中医药科技,2001,8(1):30-33
2张涛,李力,阮金兰.马鞭草化学成分对大鼠离体子宫平滑肌条作用的研究.中国中医药科技,2001,8(5):313-315
3卫生部药品标准中药成方制剂.第六册,第九册,第十九册. 1994
4国家药典委员会.中华人民共和国药典2005年版.二部.北京:化学工业出版社, 2005:35
1钟大放.以加权最小二乘法建立生物分析标准曲线的若干问题.药物分析杂志, 1996, 16(5): 343~346
2 Guidance for Industry: Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, CDER, Rockville, USA, May 2001.
3国家药典委员会.中华人民共和国药典2005年版.二部.北京:化学工业出版社, 2005:附录173
4苗志勃.验方马鞭草治疗顽固性偏头痛25例.辽宁中医杂志,2006, 33(4): 479
1刘睿,谢跃生,潘桂湘,等.药物血浆蛋白结合率测定方法的研究进展.天津中医药, 2007, 24(6):526
2张均田.药物与血浆蛋白结合的研究方法.现代药理实验方法,1669~1672
3姜纪荣,束汉麟,许国英,等.我国人和动物的磺胺药血浆蛋白结合率.药学通报, 1981,16(22):705
4徐叔云,卞如濂,陈修主编.药理学实验方法.北京:人民卫生出版社,1982,441~445
1 Jun Chen, Huimin Lin, Ming Hu. Metabolism of flavonoids via enteric recycling: Role of intestinal disposition. J. Pharmacol Exp Ther, 2003, 304(3): 1228
2 Yong Liu, ph. D. Yan Liu, et al. Enteric Disposition and Recycling of Flavonoids and Ginkgo Flavonoids. J. Alter Complement med, 2003, 9 (5): 631
3徐晶芬,卢小东,焦中秀等.马鞭草抗早孕作用机理的初步研究[J].南京医科大学学报,1998, 18(5):402-406.
4 Argento A, Tiraferri E, Marzaloni M. Oral anticoagulants and medicinal plants. An emerging interaction. Ann Ital Med Int, 2000,15(2): 139-143
1药典委员会.中华人民共和国药典2005年版一部.北京:化学工业出版社,2005
2 L. Bourdier. Verbenalin, a new glucoside derived from Verbena officinalis, J. Pharm. Chim 1908, 27(6), 49-57, 101-12
3 Horst Rimpler, Bernd Schaefer. Hastatoside, a new iridoid from Verbena officinalis and Verbena hastate. Tetrahedron Letters. 1973. 17. 1463-4
4张涛,阮金兰,吕子敏.马鞭草的化学成分研究,中国中药杂志,2000, 25(11) : 676-678
5田菁,赵毅民,栾新慧.马鞭草化学成份的研究(Ⅱ).天然产物研究与开发,2007,19:247-249
6 A. Bianco, M. Guiso, P. Passacantilli. Iridoid and phenylpropanoid glycosides from new sources, J. Nat. Prod., 1984, 47(5), 901-2
7 M. F. Lahloub, O. M. Salama, E. S. Mansour. Phenyl propanoid and iridoid glycosides from Verbena officinalis L. herb growing in Egypt, Bull. Fac. Pharm., 1990, 28(2), 75-7
8辛菲,金艺淑,沙沂,徐伟,祝峥,李玉山.马鞭草化学成分研究.中国现代中药,2008,10(10):21-25
9 Joel Reynaud, Andree Couble, Jean Raynaud. Flavonoids of Verbena officinalis L. (Verbenaceae), J. Plant Physiol., 1989, 135(3), 380-1
10田菁,赵毅民,栾新慧.马鞭草化学成份的研究.中国中药杂志,2005, 30(4): 268-269
11 M. I. Calvo, A. San Julian, M. Fernandez. Identification of the major compounds in extracts of Verbena officinalis by HPLC with post-column derivatization, Chromatographia, 1997, 46(5/6) : 241-244
12 S. A. Kawashty, I. A. El-Garf. The flavonoid chemosystematics of Egyptian Verbena species, Biochem. Syst. Ecol., 2000, 28(9) : 919-921
13 J. Reynaud, A. Couble, J. Raynaud. O-Glycosylflavonoids of Verbena officinalis L., Pharm. Acta Helv., 1992, 67(8), 216-17
14 A. D. Kawashty, LA.EI Garf. The Flavonoid chemosystematics of Egyptian species. Biochemical systematic and ecology, 2000, 28:919-921
15陈改敏,张建业,张向沛,刘宏民.马鞭草黄酮类化学成分的研究.中药材,2006,29(7): 677-679
16汤树良译.马鞭草中促进神经生长因子介导的轴突生长的新成分littorachalcone(英).国外医学中医中药分册,2004,26(3):176
17 Jose Luis Guil, Maria Esperanza Torija, Juan Jose Gimnez, Ignacio Rodriguez-Garcia. Identification of tatty acids in edible wild plants by gas chromatography, J. Chromatogr. A, 1996, 719(1), 229-35
18 Jose Luis Gui1, Maria Esperanza Torija, Juan Jose Gimenez, Ignacio Rodriguez-Garcia, Antonio Gimenez. Oxalic Acid and Calcium Determination in Wild Edible Plants, J. Agric. Food Chem., 1996, 44(7), 1821-1823
19 Mundkinajeddu Deepak, Sukhdev Swami Handa. Antiinflammatory activity and chemical composition of extracts of Verbena officinalis, Phytother. Res., 20(10, 14(6) : 463-465
20肖培根.新编中药志(第三卷),2002,第1版,32
21 Jean Cheymol. Presence of stachyose in the stems and roots of Verbena officinalis L. and the underground parts of Verbena venosa Gill and Hook. J. Pharm. Chim.,1937, 19, 110-17
22 Andree Darnat, Andra-Paul Carnat, Olivier Chavignon, Annie Heitz, Renee Wylde, Jean-Louis Lamaison. Luteolin 7-diglucuronide, the major flavonoid compound from Aloysia triphylla and Verbena officinalis, Planta Med., 1995, 61(5), 490
23桂承会,唐人九.马鞭草镇咳有效成分的研究,中药通报,1985, 10(10), 35
24 A. M. Makboul. Chemical constituents of Verbena officinalis, Fitoterapia, 1986, 57(1),50-1
25刘宏民,鮑峰玉,阎学斌.马鞭草化学成分的研究.中草药,2002, 33(6), 492-494
26 Jean-Claude Chalchat, Raymond-Philippe Garry. Chemical composition of the leaf oil of Verbena officinalis L., J. Essent. Oil Res,1996, 8(4), 419-420
27 Jean-Claude Chalchat, Raymond-Philippe Garry. Chemical composition of the leaf oil of Verbena Officinalis L. J. Essent. Oil Res, 1996, 8(4): 419-420.
28 Mohammadreza Shams Ardakani, Mahmoud Mosaddeghb, Alireza Shafaatic. Volatile Constituents from the Aerial Parts of Verbena Officinalis L. Iranian Journal of Pharmaceutical Research, 2003,39-42.
29杨再波.顶空萃取-气相色谱-质谱法分析马鞭草的挥发油组分.理化检验-化学分析,2008,44(6):514-516
30 Mundkinajeddu Deepak, Sukhdev S. Handa. 3α, 24-dihydroxy-urs-12-en-28-oic acid from Verbena Officinalis. Phytochemistry, 1998, 49(1): 269-271
31 J. L. Guil Guerrero, J. J. Gimnez Martlnez, M. E. Torija Isasa. Mineral nutrient composition of edible wild plants, Journal of Food Composition and Analysis, 1998, 11: 322-328
32江苏新医学院.中药大辞典.上册.上海:上海人民出版社.1977. 303-305.
33徐珊,焦中秀,徐小晶,等.马鞭草醇提液对绒毛膜癌JAR细胞增殖及表皮生长因子受体表达的影响.中国药科大学学报,2000,31(4): 281-284
34冯播,徐昌芬.马鞭草C部位单体4’-甲醚-黄芩素对人绒毛膜癌细胞增殖的抑制作用.中国肿瘤生物治疗杂志,2008,15(5):444-447
35徐华娥,袁红宇,欧宁.马鞭草醇提液小剂量时能显著增加紫杉醇的抗肿瘤活性.南京医科大学学报(自然科学版),2008,10(10):1275-1278
36曹志然,戎瑞雪,王蓓,等.马鞭草提取物对荷瘤小鼠抑瘤作用的实验研究.河北职工医学院学报,2008,25(2):8～10
37中华本草.第6册.上海:上海科学技术出版社,1991. 592.
38张涛,李万,阮金兰.马鞭草化学成分对大鼠离体子宫平滑肌条作用的研究.中国中医药科技,2001, 8(5): 313
39 Mundlcinajeddu Deepak, Sukhdev S. Handa. Antiinflammatory activity and composition of extracts of verbena offcinalis. Phytotherapy Research, 2000, 14(6):463-465
40苗明三主编.法定中药药理与临床.西安:世界图书出版西安公司. 1998,177-180
41徐昌芬,卢小东,焦中秀,等.马鞭草抗早孕作用机理的初步研究.南京医科大学学报,1998,18(5):402～406
42张曙萱,王海琦,欧宁.马鞭草提取液对体外培养人早孕蜕膜细胞的影响.中国天然药物,2004,2(4):242～246
43徐昌芬,曾群,徐珊,等.马鞭草醇提液中有效部位的提取及筛选.交通医学,2003,17(5):604
44 Nancy E. Hernbndez, M. L.Tereschuk, L. R. Abdala. Antimicrobial activity of the flavonoids in mediciral plants from Taft del Valle (Tucumbn, Argentina). J. Ethnopharmacol, 2000, 73(1, 2): 317-322
45陈芝芸,项柏康,朱林喜,等. 100味中药对幽门螺旋菌抑菌作用的实验研究.时珍国药研究,1996,7(1) : 25-26
46王文佳,王平,俞琦,等.马鞭草醇提物免疫活性的初步研究.贵阳中医学院学报,2008,30(4):17～18
47王文佳,王平,俞琦,等.马鞭草醇提物对小鼠IL-2生物活性的影响.甘肃中医学院学报,2008,25(2):14～15
48汤树良译.马鞭草中促进神经生长因子介导的轴突生长的新成分Littorachalcone[英].国外医学中医药分册,2004, 26(3): 177
49肖苏萍译.沿海马鞭草中1个新的具有促进神经生长因子活性的环烯醚萜糖苷[英].国外医学中医中药分册,2004, 26(6): 353
50桂承会,唐人九.马鞭草镇咳有效成分的研究.中药通报, 1985,10(10): 35
51 Kamata Yasuhiro, Toyokawa Teesuya, Teruya Masaaki, etc. CA 2004, 140(14), 223320q
52 Hurreu Richard F. Reddy Marju, Cook James D. CA 1999, 131(8), 101615h
2桂承会,唐人九.马鞭草镇咳有效成分的研究.中药通报, 1985,10(10): 35
3陈奇.中药药理研究方法学(第1版) .北京:人民卫生出版社,1994
4徐淑云.药理实验方法学(第2版) .北京:人民卫生出版社,2002,30
5冯冰虹,苏浩冲,杨俊杰.桑白皮丙酮提取物对呼吸系统的药理作用.广东药学院学报. 2005.21(1):47～49
6王立为,刘新民,余世春,等.枇杷叶抗炎和止咳作用研究.中草药. 2004,35(2): 174～176
7金正男,朴世浩,金景姬,等.引流熊胆的抗炎免疫作用.中国药理学通报. 1994,10(2): 143～146
8沈雅琴.苦刺总生物碱的抗炎作用.中国药理学通报. 1990,6(1): 10
9谢强敏,卞如廉,杨秋火,等.艾叶油的呼吸系统药理研究I,支气管扩张、镇咳和祛痰作用.中国现代应用药学杂志. 1999,16 (4):16-19
10 Enger H. and szelenyi l,Tracheal Phenolred secretion,a new methods for secreening mucosecretolytic compounds,of Pharmacological methods,Elsevier science Publishing Co,Newyork,1984,151
11周小江,曾南,贾敏如肺经草止咳化痰有效部位的初步筛选.中国民族民间医药杂志.2005,72: 46-49
12陈奇.中药药理研究方法学(第一版).人民卫生出版社,2000,7:651-652
13滕赞,李智.滨篙内醋对气管平滑肌的舒张作用及其细胞内钙的影响,2005,5: 13
14赵明沂,李智,丛华,等.滨篙内酷对豚鼠离体气管平滑肌张力的影响.中国药理学会通讯,2000,17(2):64
15潘泽等.国外疾学-内科学分册. 1999,26(3):11
1唐人九.人字草黄酮类化学成分研究.华西药学杂志,1996,11(1) :51
2喻蓉,许庆,李伯刚,等.搭棚藤的化学成分研究.天然产物研究与开发,2003,15(5) :4051
3许凤鸣,王兆全,李有文.半枝莲化学成分的研究.中国现代应用药学,1997,14(6) :81
4田菁,赵毅民,栾新慧.马鞭草化学成份的研究(Ⅱ).天然产物研究与开发, 2007,19:247-249
5陈改敏,张建业,张向沛,刘宏民.马鞭草黄酮类化学成分的研究.中药材,2006,29(7): 677-679
6于德泉,杨竣山.分析化学手册第二版.第七分册.核磁共振波谱分析.北京:化学工业出版社,1999,818,820
7蒋受军,朱彬,魏锋,等.小野芝麻化学成分的研究(I).中国中药杂志,2002, 27(9): 671-673
8 A. Bianco, M. Guiso, P. Passacantilli. Iridoid and phenylpropanoid glycosides from new sources. J. Nat. Prod., 1984, 47(5): 901-2
9 M. F. Lahloub, O. M. Salama, E. S. Mansour. Phenyl propanoid and iridoid glycosides from Verbena officinalis L. herb growing in Egypt. Bull. Fac. Pharm., 1990, 28(2): 75-7
10桂承会,唐人九.马鞭草镇咳成分的研究.中药通报,1985,10(10) :351
11 Dirk Teborg, Peter Junio. Iridoid glucosides from Penstemon nittdus. Planta Med, 1991, 57 :1841
12 Dirk Teborg, Peter Junior1 Iridoid glucosides from Penstemon nittdus. Planta Med , 1991, 57 :1841
1张涛,谢虹,李万.马鞭草提取物抗大鼠早孕作用的研究.中国中医药科技,2001,8(1):30-33
2张涛,李力,阮金兰.马鞭草化学成分对大鼠离体子宫平滑肌条作用的研究.中国中医药科技,2001,8(5):313-315
3卫生部药品标准中药成方制剂.第六册,第九册,第十九册. 1994
4国家药典委员会.中华人民共和国药典2005年版.二部.北京:化学工业出版社, 2005:35
1钟大放.以加权最小二乘法建立生物分析标准曲线的若干问题.药物分析杂志, 1996, 16(5): 343~346
2 Guidance for Industry: Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, CDER, Rockville, USA, May 2001.
3国家药典委员会.中华人民共和国药典2005年版.二部.北京:化学工业出版社, 2005:附录173
4苗志勃.验方马鞭草治疗顽固性偏头痛25例.辽宁中医杂志,2006, 33(4): 479
1刘睿,谢跃生,潘桂湘,等.药物血浆蛋白结合率测定方法的研究进展.天津中医药, 2007, 24(6):526
2张均田.药物与血浆蛋白结合的研究方法.现代药理实验方法,1669~1672
3姜纪荣,束汉麟,许国英,等.我国人和动物的磺胺药血浆蛋白结合率.药学通报, 1981,16(22):705
4徐叔云,卞如濂,陈修主编.药理学实验方法.北京:人民卫生出版社,1982,441~445
1 Jun Chen, Huimin Lin, Ming Hu. Metabolism of flavonoids via enteric recycling: Role of intestinal disposition. J. Pharmacol Exp Ther, 2003, 304(3): 1228
2 Yong Liu, ph. D. Yan Liu, et al. Enteric Disposition and Recycling of Flavonoids and Ginkgo Flavonoids. J. Alter Complement med, 2003, 9 (5): 631
3徐晶芬,卢小东,焦中秀等.马鞭草抗早孕作用机理的初步研究[J].南京医科大学学报,1998, 18(5):402-406.
4 Argento A, Tiraferri E, Marzaloni M. Oral anticoagulants and medicinal plants. An emerging interaction. Ann Ital Med Int, 2000,15(2): 139-143
1药典委员会.中华人民共和国药典2005年版一部.北京:化学工业出版社,2005
2 L. Bourdier. Verbenalin, a new glucoside derived from Verbena officinalis, J. Pharm. Chim 1908, 27(6), 49-57, 101-12
3 Horst Rimpler, Bernd Schaefer. Hastatoside, a new iridoid from Verbena officinalis and Verbena hastate. Tetrahedron Letters. 1973. 17. 1463-4
4张涛,阮金兰,吕子敏.马鞭草的化学成分研究,中国中药杂志,2000, 25(11) : 676-678
5田菁,赵毅民,栾新慧.马鞭草化学成份的研究(Ⅱ).天然产物研究与开发,2007,19:247-249
6 A. Bianco, M. Guiso, P. Passacantilli. Iridoid and phenylpropanoid glycosides from new sources, J. Nat. Prod., 1984, 47(5), 901-2
7 M. F. Lahloub, O. M. Salama, E. S. Mansour. Phenyl propanoid and iridoid glycosides from Verbena officinalis L. herb growing in Egypt, Bull. Fac. Pharm., 1990, 28(2), 75-7
8辛菲,金艺淑,沙沂,徐伟,祝峥,李玉山.马鞭草化学成分研究.中国现代中药,2008,10(10):21-25
9 Joel Reynaud, Andree Couble, Jean Raynaud. Flavonoids of Verbena officinalis L. (Verbenaceae), J. Plant Physiol., 1989, 135(3), 380-1
10田菁,赵毅民,栾新慧.马鞭草化学成份的研究.中国中药杂志,2005, 30(4): 268-269
11 M. I. Calvo, A. San Julian, M. Fernandez. Identification of the major compounds in extracts of Verbena officinalis by HPLC with post-column derivatization, Chromatographia, 1997, 46(5/6) : 241-244
12 S. A. Kawashty, I. A. El-Garf. The flavonoid chemosystematics of Egyptian Verbena species, Biochem. Syst. Ecol., 2000, 28(9) : 919-921
13 J. Reynaud, A. Couble, J. Raynaud. O-Glycosylflavonoids of Verbena officinalis L., Pharm. Acta Helv., 1992, 67(8), 216-17
14 A. D. Kawashty, LA.EI Garf. The Flavonoid chemosystematics of Egyptian species. Biochemical systematic and ecology, 2000, 28:919-921
15陈改敏,张建业,张向沛,刘宏民.马鞭草黄酮类化学成分的研究.中药材,2006,29(7): 677-679
16汤树良译.马鞭草中促进神经生长因子介导的轴突生长的新成分littorachalcone(英).国外医学中医中药分册,2004,26(3):176
17 Jose Luis Guil, Maria Esperanza Torija, Juan Jose Gimnez, Ignacio Rodriguez-Garcia. Identification of tatty acids in edible wild plants by gas chromatography, J. Chromatogr. A, 1996, 719(1), 229-35
18 Jose Luis Gui1, Maria Esperanza Torija, Juan Jose Gimenez, Ignacio Rodriguez-Garcia, Antonio Gimenez. Oxalic Acid and Calcium Determination in Wild Edible Plants, J. Agric. Food Chem., 1996, 44(7), 1821-1823
19 Mundkinajeddu Deepak, Sukhdev Swami Handa. Antiinflammatory activity and chemical composition of extracts of Verbena officinalis, Phytother. Res., 20(10, 14(6) : 463-465
20肖培根.新编中药志(第三卷),2002,第1版,32
21 Jean Cheymol. Presence of stachyose in the stems and roots of Verbena officinalis L. and the underground parts of Verbena venosa Gill and Hook. J. Pharm. Chim.,1937, 19, 110-17
22 Andree Darnat, Andra-Paul Carnat, Olivier Chavignon, Annie Heitz, Renee Wylde, Jean-Louis Lamaison. Luteolin 7-diglucuronide, the major flavonoid compound from Aloysia triphylla and Verbena officinalis, Planta Med., 1995, 61(5), 490
23桂承会,唐人九.马鞭草镇咳有效成分的研究,中药通报,1985, 10(10), 35
24 A. M. Makboul. Chemical constituents of Verbena officinalis, Fitoterapia, 1986, 57(1),50-1
25刘宏民,鮑峰玉,阎学斌.马鞭草化学成分的研究.中草药,2002, 33(6), 492-494
26 Jean-Claude Chalchat, Raymond-Philippe Garry. Chemical composition of the leaf oil of Verbena officinalis L., J. Essent. Oil Res,1996, 8(4), 419-420
27 Jean-Claude Chalchat, Raymond-Philippe Garry. Chemical composition of the leaf oil of Verbena Officinalis L. J. Essent. Oil Res, 1996, 8(4): 419-420.
28 Mohammadreza Shams Ardakani, Mahmoud Mosaddeghb, Alireza Shafaatic. Volatile Constituents from the Aerial Parts of Verbena Officinalis L. Iranian Journal of Pharmaceutical Research, 2003,39-42.
29杨再波.顶空萃取-气相色谱-质谱法分析马鞭草的挥发油组分.理化检验-化学分析,2008,44(6):514-516
30 Mundkinajeddu Deepak, Sukhdev S. Handa. 3α, 24-dihydroxy-urs-12-en-28-oic acid from Verbena Officinalis. Phytochemistry, 1998, 49(1): 269-271
31 J. L. Guil Guerrero, J. J. Gimnez Martlnez, M. E. Torija Isasa. Mineral nutrient composition of edible wild plants, Journal of Food Composition and Analysis, 1998, 11: 322-328
32江苏新医学院.中药大辞典.上册.上海:上海人民出版社.1977. 303-305.
33徐珊,焦中秀,徐小晶,等.马鞭草醇提液对绒毛膜癌JAR细胞增殖及表皮生长因子受体表达的影响.中国药科大学学报,2000,31(4): 281-284
34冯播,徐昌芬.马鞭草C部位单体4’-甲醚-黄芩素对人绒毛膜癌细胞增殖的抑制作用.中国肿瘤生物治疗杂志,2008,15(5):444-447
35徐华娥,袁红宇,欧宁.马鞭草醇提液小剂量时能显著增加紫杉醇的抗肿瘤活性.南京医科大学学报(自然科学版),2008,10(10):1275-1278
36曹志然,戎瑞雪,王蓓,等.马鞭草提取物对荷瘤小鼠抑瘤作用的实验研究.河北职工医学院学报,2008,25(2):8～10
37中华本草.第6册.上海:上海科学技术出版社,1991. 592.
38张涛,李万,阮金兰.马鞭草化学成分对大鼠离体子宫平滑肌条作用的研究.中国中医药科技,2001, 8(5): 313
39 Mundlcinajeddu Deepak, Sukhdev S. Handa. Antiinflammatory activity and composition of extracts of verbena offcinalis. Phytotherapy Research, 2000, 14(6):463-465
40苗明三主编.法定中药药理与临床.西安:世界图书出版西安公司. 1998,177-180
41徐昌芬,卢小东,焦中秀,等.马鞭草抗早孕作用机理的初步研究.南京医科大学学报,1998,18(5):402～406
42张曙萱,王海琦,欧宁.马鞭草提取液对体外培养人早孕蜕膜细胞的影响.中国天然药物,2004,2(4):242～246
43徐昌芬,曾群,徐珊,等.马鞭草醇提液中有效部位的提取及筛选.交通医学,2003,17(5):604
44 Nancy E. Hernbndez, M. L.Tereschuk, L. R. Abdala. Antimicrobial activity of the flavonoids in mediciral plants from Taft del Valle (Tucumbn, Argentina). J. Ethnopharmacol, 2000, 73(1, 2): 317-322
45陈芝芸,项柏康,朱林喜,等. 100味中药对幽门螺旋菌抑菌作用的实验研究.时珍国药研究,1996,7(1) : 25-26
46王文佳,王平,俞琦,等.马鞭草醇提物免疫活性的初步研究.贵阳中医学院学报,2008,30(4):17～18
47王文佳,王平,俞琦,等.马鞭草醇提物对小鼠IL-2生物活性的影响.甘肃中医学院学报,2008,25(2):14～15
48汤树良译.马鞭草中促进神经生长因子介导的轴突生长的新成分Littorachalcone[英].国外医学中医药分册,2004, 26(3): 177
49肖苏萍译.沿海马鞭草中1个新的具有促进神经生长因子活性的环烯醚萜糖苷[英].国外医学中医中药分册,2004, 26(6): 353
50桂承会,唐人九.马鞭草镇咳有效成分的研究.中药通报, 1985,10(10): 35
51 Kamata Yasuhiro, Toyokawa Teesuya, Teruya Masaaki, etc. CA 2004, 140(14), 223320q
52 Hurreu Richard F. Reddy Marju, Cook James D. CA 1999, 131(8), 101615h