摘要
冬凌草为唇形科香茶菜属植物碎米桠(Isodon rubescens(Hemsl.) Hara)的干燥地上部分,以草质部分入药。广泛分布于我国黄河长江流域,主产于河南济源太行山一带。味甘苦,性微寒,具有清热解毒、消炎止痛、健胃活血及抗肿瘤之功效。民间常常用于治疗咽喉肿痛、扁桃体炎、蛇虫咬伤,并对食管癌、贲门癌、原发性肝癌、肺癌、前列腺癌、膀胱癌等有一定疗效,对食管上皮增生有显著疗效。植物研究证实冬凌草含有二萜、黄酮、酚酸、三萜和挥发油等化学成分。目前已有冬凌草用于抗癌的片剂和治疗咽痛的含片应用于临床。二萜是冬凌草中一种主要的药效活性成分,但由于种类多样且含量较低,使其深入研究受到限制。然而对于冬凌草的大量研究仅处在提取分离阶段,而对其质量控制方法和药物代谢动力学方面的研究却很少。
本文通过对冬凌草进行系统分离,确定其中的化学成分,采用LC-MS作为主要分析方法,利用其快速高效、灵敏度高、特异性强的特点,对冬凌草中的微量和痕量物质进行定性和定量分析,并推断其中的未知成分。同时研究二萜类成分在动物体内的过程和代谢规律研究,为进一步开发利用二萜类有效成分提供技术支持。
第一部分冬凌草化学成分的研究
目的:本研究以冬凌草乙酸乙酯和正丁醇部分为研究对象,利用硅胶柱色谱、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系统分离研究,并应用MS、1~H-NMR、(13)~C-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:干燥的冬凌草(10 Kg)适当粉碎后,用95%乙醇冷浸3次,合并浸出液加热,加入活性炭趁热抽滤,滤液减压浓缩至粘膏状(340 g),将其悬浮于饱和NaCl水溶液中,分别用石油醚、三氯甲烷、乙酸乙酯和正丁醇萃取,继而得到石油醚部位(8 g)、三氯甲烷部位(32 g)和乙酸乙酯部位(48 g)、正丁醇部位(36 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、葡聚糖凝胶柱色谱,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1~H-NMR、(13)~C-NMR波谱学方法对分离得到的单体化合物进行结构确证。
结果:冬凌草通过系统提取分离,从中得到7个单体化合物,其中包括1个二萜类化合物,5个酚酸,1个甾醇:冬凌草甲素,咖啡酸,阿魏酸,原儿茶醛,水杨酸,绿原酸,β-谷甾醇。
结论:本文对冬凌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、葡聚糖凝胶柱色谱、制备薄层色谱、制备液相色谱等分离手段,共分离得到7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确证,其中包括1个二萜类化合物,5个酚酸,1个甾醇:冬凌草甲素,咖啡酸,阿魏酸,原儿茶醛,水杨酸,绿原酸,β-谷甾醇,其中绿原酸为首次从该植物中提取分离得到。
第二部分液质联用技术鉴定冬凌草中的二萜类成分
目的:总结贝壳杉烷型二萜类成分的电喷雾质谱裂解规律,建立灵敏、有效的可用于鉴定冬凌草中二萜类成分的液质联用(LC-MS)分析方法。
方法:通过电喷雾离子源负离子一级质谱扫描、母离子扫描和子离子扫描,研究16个二萜类成分对照品的质谱裂解途径,并总结其规律,进而联合采用多离子监测-信息依赖-增强型子离子扫描(MIM-IDA-EPI)和中性粒子缺失-信息依赖-增强型子离子扫描(NL-IDA-EPI)两种模式分析鉴定冬凌草中二萜类化合物。采用C18柱进行色谱分离,流动相为0.1%甲酸水-甲醇,梯度洗脱。
结果:总结出7个二萜类成分的质谱裂解规律和相同构型且含有相同取代基的同分异构体在RP-HPLC上的洗脱顺序规律。据此鉴定出冬凌草提取物中40个二萜类成分。
结论:本法灵敏度高、专属性强,成功用于冬凌草中二萜类化合物的结构鉴定。实验证实,MIM-IDA-EPI和NL-IDA-EPI扫描模式联用技术在化合物结构分析方面具有超过其单一模式的优越性,对控制中药材质量具有重要意义。
第三部分液质联用技术同时测定冬凌草中28种成分及冬凌草质量表征
目的:建立一种快速、准确、可同时测定冬凌草中28种化学成分(19种二萜类成分、6种酚酸及3种黄酮)的HPLC-ESI-MS分析方法,并用于冬凌草药材分析及质量调查。
方法:取冬凌草药材粉末约1.0g,精密称定,加入25mL甲醇,密塞,称定重量,超声处理60min,放冷,称重,用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。由MRM-IDA-EPI模式对28种化合物结构进行确证,采用MRM正负离子扫描模式建立其含量测定方法,并采用所建立的方法对不同产地的21批冬凌草药材进行分析。质谱条件:ESI源;正负离子同时监测;源喷射电压(IS):5500V(正离子监测模式),-4500 V(负离子监测模式);离子源温度:600℃。雾化气(Gas1):40psi,加热气(Gas2):50psi,帘气:25psi。色谱条件:色谱柱:Diamonsil C18 column(250mm×4.6mm,5μm);柱温:25℃;流动相:0.1%甲酸-甲醇(含0.1%甲酸),梯度洗脱;分析时间:15.1min;流速:0.7mL·min~(-1),进样量10μL。
结果:28种化学成分在测定浓度范围内均具有良好的线性关系,精密度、准确度、检测限和定量限均符合要求。样品测定结果表明,冬凌草产地、生长环境和采集时间均对28种活性成分的含量有影响,其中产地时间和生长环境是其中较为重要的影响因素。
结论:本法简便、快速、专属性好,可用于冬凌草中28种化学成分的含量测定,除冬凌草甲素和冬凌草乙素外,其他17种二萜均为首次测定。该法对于冬凌草药材的质量控制具有重要意义。
第四部分液质联用技术同时测定大鼠血浆中拉西多宁、冬凌草甲素、冬凌草乙素和牛尾草素A及其药代动力学研究
目的:建立一种基于多组分同时测定大鼠血浆中拉西多宁、冬凌草甲素、冬凌草乙素和牛尾草素A含量的HPLC-MS方法,并用于大鼠灌胃给予冬凌草提取物后该4种成分的药代动力学研究,建立其药-时曲线,阐明药动学参数与特征。
方法:大鼠灌胃给与冬凌草提取物(10mL·kg~(-1))后,分别于给药后10, 30,60,90,105,120,150,180,210,240,300和540min眼内眦静脉丛取血,制备血浆样品,采用液-液提取方法进行样品预处理,磺胺甲恶唑为内标。色谱柱为C18柱,流动相为0.1%甲酸水-甲醇,梯度洗脱,运行时间为13.50min。采用电喷雾离子源(ESI),多周期正负离子同时扫描,多反应监测(MRM)模式进行定量。4种被测成分的监测离子对分别为拉西多宁和冬凌草甲素m/z 365.3/347.3,冬凌草乙素m/z 361.2/343.2,牛尾草素A m/z 363.2/283.1和磺胺甲恶唑m/z 254.1/156.0。
结果:血浆中拉西多宁、冬凌草甲素、冬凌草乙素和牛尾草素A分别在2.24～2240、4.92～4920、5.32～5320和1.36～1360ng·mL~(-1)范围内线性关系良好(r≥0.998),最低定量限(LLOQ)≤1.36ng·mL~(-1)。日内、日间精密度的相对标准偏差(RSD)均小于9.1%,相对误差(RE)为-8.1%～4.1%。平均提取回收率为70.2%～108.0%。大鼠单次灌胃冬凌草提取物后,血浆中4种成分的药代动力学参数t_(1/2α),t_(1/2β)和MRT具有一致性,且4种成分的吸收和消除均较快,但拉西多宁、冬凌草甲素和牛尾草素A的达峰时间比冬凌草乙素达峰时间长。
结论:该法灵敏度高、选择性好、精密度好,可用于大鼠灌胃给与冬凌草提取物后,血浆中拉西多宁、冬凌草甲素、冬凌草乙素和牛尾草素A的药代动力学研究,该结果可为冬凌草临床用药提供参考。
第五部分液质联用技术测定大鼠胆汁中10种二萜类成分及其在胆汁中的排泄研究
目的:建立一种可同时测定大鼠胆汁中10种主要二萜(开展香茶菜甲素,拉西多宁,冬凌草甲素,表诺多星,诺多星,冬凌草乙素,牛尾草素A,延命草醇,毛栲利素和贝壳松醇)含量的HPLC-MS方法,并用于大鼠灌胃给予冬凌草提取物后该10种成分的胆汁排泄研究。
方法:健康SD雄性大鼠8只,随机分为空白对照组(2只)和试验组(6只)。试验组大鼠灌胃给予冬凌草提取物(10mL·kg~(-1)),空白对照组给予等量的水后,行胆管切开术。分别收集0-1h,1-3h,3-5h,5-8h,8-12 h,12-20h和20-24h时间段的胆汁样品。采用液-液提取方法进行样品前处理,磺胺甲恶唑为内标。色谱柱为C18柱,流动相为0.1%甲酸水-甲醇,梯度洗脱,运行时间为15.10min。采用电喷雾离子源(ESI),正负离子扫描,多反应监测(MRM)模式进行定量。测定大鼠灌胃给予冬凌草提取物后胆汁中10种二萜的累积排泄率。
结果:所测的10种二萜中开展香茶菜甲素,拉西多宁,冬凌草甲素,表诺多星和冬凌草乙素的累积排泄率分别占给药剂量的10.67,11.23,9.77,20.81和8.55%,而化合物诺多星,牛尾草素A,延命草醇,毛栲利素和贝壳松醇的累积排泄率分别占给药剂量的20.61,17.22,15.63,8.87和19.42%。
结论:经方法学考察符合生物样品的测定要求,可用于大鼠胆汁中10种二萜浓度的测定及排泄研究,待测10种二萜在0~1h内,排泄率最高,随着时间的推移,排泄率逐渐减少,但在3~5h时间段时略有增加,这可能是由于上述成分在肠道不同部位有所吸收入血所致,随后再逐渐下降所致。
第六部分冬凌草甲素不同血浆中蛋白结合率测定
目的:建立冬凌草甲素在大鼠血浆、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:冬凌草甲素的血浆蛋白结合率分别为:大鼠血浆:69.66±12.8%、59.62±12.6%、57.94±4.1%;人血浆:78.15±3.6%、77.92±8.8%、76.72±7.3%;牛血清白蛋白:35.58±7.2%、34.59±10.8%、32.03±6.0%。
结论:本文应用平衡透析法研究了冬凌草甲素与人血浆蛋白,牛血清白蛋白和大鼠血浆蛋白的结合情况,结果表明冬凌草甲素的血浆蛋白结合率在60%~70%之间,属中度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,并且不具有质量浓度依赖性,不易引起具有药理作用的游离型血药质量浓度发生明显变化,说明冬凌草甲素临床用药有较好的安全性。
第七部分冬凌草甲素大鼠在体肠吸收动力学研究
目的:建立同时测定肠循环液中冬凌草甲素与酚红浓度的HPLC/DAD法,探讨冬凌草甲素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(250mm×4.6mm,5μm);柱温30℃;流动相为甲醇-乙腈-0.5%的磷酸(55:15:30),流速1.0 mL·min~(-1);检测波长238nm;进样量20μL。
结果:在5.0～15μg·mL~(-1)内冬凌草甲素的吸收量与质量浓度成线性关系,Ka值基本保持不变;冬凌草甲素各肠段的吸收速率无显著性差异,冬凌草甲素在十二指肠、空肠、回肠、结肠的Ka值分别为(0.0475±0.0062),(0.0468±0.0051),(0.0346±0.0037),(0.0435±0.0023)h~(-1)。
结论:本文首次建立了HPLC/DAD法同时测定肠循环液中冬凌草甲素及酚红的浓度,该法操作简便,结果准确,灵敏度高。研究结果表明,冬凌草甲素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;冬凌草甲素在整个肠道均有吸收,提示可以将冬凌草甲素研制成缓释制剂。
Isodon rubescens (Hemsl.) Hara (labiatae) is a perennial herb that is native to the Yellow River valley and the Yangtze River in China. Isodon rubescens (formally named Rabdosia rubescens) is the entire dried plant of R. rubescens (Hemsl) Hara. It is a well-known traditional Chinese medicine (TCM), called "Dong Ling Cao" in Chinese. This herb has long been used as a folk remedy for respiratory and gastrointestinal bacterial infections, inflammation, and cancer. Phytochemical studies of Isodon rubescens have revealed that it contains diterpenoids, flavonoids, phenolic acids, triterpenoids and volatile oils, as well as other chemicals. Some preparations that are extracted from Isodon rubescens have existed for a long time, including anti-cancer tablets and buccal tablets for sore throat treatment. It has been reported that diterpenoids are the most important active constituents that contribute to the pharmacological efficacy of Isodon rubescens. However, for a large number of studies only in Isodon rubescens extraction and separation stages, and its quality control methods and pharmacokinetics of research are few.
In this paper, the main components was isolated and purified in Isodon rubescens. HPLC-MS was performed to analyze chemical components in Isodon rubescens extract and biological samples. By HPLC-MS method, diterpenoids were characterized in crude extract of Isodon rubescens with the summarized fragmentation rules. Meanwhile, a novel sensitive and selective HPLC-ESI-MS/MS method was developed and validated to simultaneously determinate and identify constituents in Isodon rubescens samples. Then, a sensitive and selective LC-ESI-MS method for the simultaneous determination of diterpenoids in rat plasma was firstly developed and validated to analyze plasma samples of the four analytes after oral administration of Isodon rubescens. Finally, a HPLC-MS method was established for the quantification of diterpenoids in rat bile after oral administration of Isodon rubescens extract. At the same time, drug-protein binding and absorption kinetics of oridonin were studies. These results of this work will contribution to its development and utilization.
Part one Studies on the chemical constituents of Isodon rubescens
Objective: To isolate and purify the compounds in Isodon rubescens. by the technologies of Silica gel column chromatography, Sephadex LH-20 column chromatography, preparative TLC, HPLC and recrystallization and to characterize the structures of the isolated pure compounds by using the spectroscopic methods including MS, 1~H-NMR, (13)~C-NMR.
Methods: The pulverized dried aerial parts of Isodon rubescens (10 Kg) were macerated for 7 days at room temperature with 95% alcohol for three 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 crud extract of 340 g. Then, 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 8 g, the trichloromethane fraction 32 g, the ethyl acetate fraction 48 g and the n-butyl alcohol fraction 36 g. The ethyl acetate fraction and n-butyl alcohol fraction were applied to 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, preparative TLC and 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: 7 compounds were yielded by systemical separation of the aerial parts of Isodon rubescens. The structures of 7 compounds were identified on the basis of chemical and spectral analysis, including 1 diterpenoid, 5 phenolic acids and a phytostero. They were oridonin (1), caffeic acid (2), ferulic acid (3), protocatechuic aldehyde (4), salicylic acid (5), chlorogenic acid (6), β-sitosterol (7).
Conclusion: The result indicated that the solvents and methods of extraction and isolation used in this experiment were practicable. Silica gel column chromatography, preparative TLC and HPLC were employed to isolate and purify the components of the aerial parts of Isodon rubescens, and spectroscopic methods were used to establish the structures of the compounds.
7 compounds were obtained and identified with the aid of spectroscopic methods and chlorogenic acid was fistly obtained in this herb.
Part two Screening and characterization of diterpenoids in Isodon rubescens using liquid chromatography coupled with hybrid triple quadruple linear ion trap mass spectrometry
Objective: To summarize fragmentation rules and develop a high sensitive and efficient liquid chromatography-mass spectrometry (LC-MS) method for detection and characterization of the trace diterpenoids in Isodon rubescens.
Methods: First, we studied the mass fragmentation patterns of 16 diterpenoids standards in the negative ion mode by full scan mass spectra, product ion scan (PI) and precursor ion scan (PREC). On the basis of the summarized new rules, diterpenoids in a crude extract of Isodon rubescens were characterized by the combined use of the MIM-IDA-EPI and NL-IDA-EPI modes on a hybrid triple quadrupole-linear ion trap mass spectrometer, for the first time. The chromatographic conditions: Diamonsil C18 column (250mm×4.6mm, 5μm), and the column temperature was kept at room temperature. The mobile phase was composed of 0.1% aqueous formic acid (A) -method (B) with gradient elution.
Results: Seven fragmentation rules were summarized. According to the summarized fragmentation rules, polarity rules of isomer, retention times, accurate molecular weights and characteristic fragment ions, 40 diterpenoids in crude extract of Isodon rubescens were characterized.
Conclusion: In this study, a high sensitive, accurate and effective LC- MS method for on-line qualitative analysis of the trace diterpenoids in Isodon rubescens has been developed. This study has demonstrated the unprecedented advantage of the combination use of the MIM-IDA-EPI and NL-IDA-EPI mode. The MIM-IDA-EPI mode is sensitive and no pre-acquisition of MS/MS spectra of the parent ion due to the same precursor ion and product ion. Therefore, the characterization of the trace diterpenoids has become very easy and accurate by combination use of the two modes. It has played an important role in controlling the quality of medicinal herb and supplied a method for other trace diterpenoids compounds from natural and synthetic source in the future.
Part three Simultaneous qualitative and quantitative analysis of 28 components in Isodon rubescens by high performance liquid chromatography-electrospray ionization tandem mass spectrometry
Objective: To develop a novel qualitative and quantitative method using high performance liquid chromatography coupled with tandem mass spectrometry for simultaneous analysis of 28 components including 19 diterpenoids, 6 phenolic acids and 3 flavonoids in Isodon rubescens, an important traditional Chinese medicine.
Methods: First, an information-dependent acquisition (IDA) method was employed to trigger product ion scans above the MRM signal threshold so that the 28 components could be identified through enhanced product ion (EPI) scans. Second, multiple-reaction monitoring (MRM) was employed in positive and negative mode at the same time in single analysis process and validated to simultaneously determinate and identify 28 constituents in 21 batches of Isodon rubescens. The instrument operated using electrospray ionization source in positive and negative mode simultaneously. The ion spray voltage was set to 5500V and -4500V, respectively. The turbo spray temperature was maintained at 600°C. Nebulizer gas (gas 1) and heater gas (gas 2) was set at 40 and 50 psi, respectively. The curtain gas was kept at 25 psi and interface heater was on. Nitrogen was used in all cases. The chromatographic separation was performed on a Diamonsil C18 column with linear gradient elution with 0.1% aqueous formic acid/methanol containing 0.1% formic acid at a flow rate of 0.7mL·min~(-1) for 15.1 min and the column temperature set at 25°C.
Results: The linear relationships, linearity, precision, accuracy, limit of detection and limit of quantification of the method were good for the 28 components. And we successfully applied it to analyze 21 Isodon rubescens samples from different sources. The results demonstrated that a number of reasons such as plant origin, growth circumstance and harvest time might contribute to the differences in the level of active constituents among various Isodon rubescens samples. Plant origin and growth circumstance were principal reasons. These all suggested the quality of Isodon rubescens could be assured if locality and collecting time should be standardized.
Conclusion: A novel sensitive and selective HPLC-ESI-MS/MS method operating both negative and positive scanning modes in single analysis process was developed and validated to simultaneously determinate and identify 28 constituents in Isodon rubescens samples. The satisfactory results demonstrated that the HPLC-MS/MS method offered a good alternative for routine analysis due to its rapidness, sensitivity and specificity and could be applied as a reliable quality evaluation method for Isodon rubescens. In the future, HPLC-MS/MS method would be more and more popular in analyzing herb medicine.
Part four Simultaneous determination of lasiodonin, oridonin, ponicidin and rabdoternin A in rat plasma by liquid chromatography-electrospray ionization mass spectrometric method and its application to pharmacokinetic study of Isodon rubescens extract
Objective: To develop a sensitive, specific and rapid liquid chromatography-mass spectrometry (LC-MS) method and validate for the simultaneous determination of lasiodonin, oridonin, ponicidin and rabdoternin A in rat plasma, using sulfamethoxazole as an internal standard (IS).
Methods: Blood samples were collected into heparinized centrifuge tubes from the fossa orbitalis vein at 10, 30, 60, 90, 105, 120, 150, 180, 210, 240, 300 and 540min after single oral administration of Isodon rubescens decoction (10mL·kg~(-1)). Within 30min after blood withdrawal, the samples were centrifuged at 4 000 rpm for 10min and the separated plasma samples were frozen in polypropylene tubes at -20°C prior to analysis. The plasma samples were pretreated by liquid–liquid extraction (LLE) with acetic ether and chromatographic separation was performed on a C18 column with linear gradient elution using water and methanol, which were both acidified with 0.1% aqueous formic acid, at a flow rate of 0.8mL·min~(-1).The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization (ESI) source operating. Higher sensitivity was achieved by setting three scanning periods in a novel detection mode. The optimized mass transition ion-pairs (m/z) for quantitation were 365.3/347.3 for lasiodonin and oridonin, 361.2/343.2 for ponicidin, 363.2/283.1 for rabdoternin A, and 254.1/156.0 for IS. The total run time was 13.50 min between injections.
Results: The calibration curves were linear over the investigated concentration range: 2.24～2240 ng·mL~(-1)(lasiodonin), 4.92～4920 ng·mL~(-1) (oridonin), 5.32～5320 ng·mL~(-1)(ponicidin) and 1.36～1360 ng·mL~(-1) (rabdoternin A), with all correlation coefficients higher than 0.998. The lower limits of quantitation (LLOQ) of these analytes were less than 1.36 ng·mL~(-1). The intra- and inter-day RSD were no more than 9.1% and the relative errors were within the range of -8.1% to 4.1%. The average extraction recoveries for all compounds were between 70.2 %-108.0%. The four analytes have parallel pharmacokinetic parameters in vivo, such as t_(1/2α), t_(1/2β) and MRT. All of the four analytes were absorbed rapidly and eliminated quickly with the similar rate. But the T_(max0 of lasiodonin, oridonin, rabdoternin A was longer than that of ponicidin.
Conclusion: A sensitive and selective LC-ESI-MS method for the simultaneous determination of lasiodonin, oridonin, ponicidin and rabdoternin A in rat plasma was firstly developed and validated. The proposed method showed appropriate accuracy and repeatability and was successfully applied to a plasma samples analysis of the four analytes after oral administration of Isodon rubescens extract, which maybe provide some references to the apprehension of the action mechanism and clinical application of Isodon rubescens.
Part five Quantitative analysis of ten diterpenoids in rat bile after oral administration of Isodon rubescens extract by high performance liquid chromatography-electrospray ionization tandem mass spectrometry
Objective: A sensitive and selective HPLC–ESI–MS/MS method for simultaneous determination of ten major diterpenoids (effusanin A, lasiodonin, oridonin, epinodosin, nodosin, ponicidin, rabdoternin A, enmenol, lasiokaurin and lasiokaurinol) in rat bile. Using this method, the biliary excretion profiles of these diterpenoids were further investigated after a single oral administration of Isodon rubescens extract.
Methods: Eight healthy rats were divided into experimental group and blank group. For experimental group, six rats were administered with Isodon rubescens extract at a single oral dosage of 10mL·kg~(-1), while the other two rats were administered with deionized water at an equal dose for blank. After these rats had been anesthetized, an abdominal incision was made and the common bile duct was cannulated with PE-10 tubing for the collection of bile samples. A heating lamp was used for maintaining the body temperature during the experimental procedure to prevent hypothermic alterations of the bile flow. Bile samples were collected during 0-1h, 1-3h, 3-5h, 5-8h, 8-12h, 12-20h and 20-24h periods. Blank bile samples were collected to check whether they were free of interfering components. All samples were stored at -20°C until additional extraction and analysis. A simple liquid-liquid extraction method was applied to extract the ten diterpenoids and IS from rat bile. The chromatographic separation was performed on a Diamonsil C18 column (250mm×4.6mm, 5μm), and the column temperature set at 25°C. A linear gradient elution of methanol and water was used for the separation. The analyses were performed using an electrospray ionization source in positive and negative mode respectively. Multiple–reaction monitoring (MRM) mode was carried out for obtaining the maximum sensitivity for the detection of the target compounds.
Results: A rapid HPLC-ESI-MS/MS method was established for the simultaneous quantification concentrations of ten diterpenoids in bile. The cumulative biliary excretion of diterpenoids (effusanin A, lasiodonin, oridonin, epinodosin, ponicidin,) over the dose administered was 10.67, 11.23, 9.77, 20.81, 8.55%, respectively, while compound (nodosin, rabdoternin A, enmenol, lasiokaurin, lasiokaurinol)was 20.61, 17.22, 15.63, 8.87, 19.42%, respectively.
Conclusion: The specificity, linearity, accuracy, precision, recovery, matrix effect and several of stabilities have been validated for diterpenoids in rat bile samples. The results showed that this method is robust, specific and sensitive and it can successfully fulfill the requirement of excretion study of the ten diterpenoids in Isodon rubescens. These amounts gradually decreased with time. At different times, bile excretion of these diterpenoids was the highest in 0-1h. These amounts gradually decreased with time. The concentration of these diterpenoids increased in 3-5h, which may be caused by these diterpenoids absorbed into the blood in different parts of the intestine, and then decreased gradually.
Part six Drug-protein binding determination of oriodonin
Objective: To develop a high performance liquid chromatography (HPLC) to determine the protein binding rates of oridonin in human plasma, rat plasma, bovine serum albumin (BSA), and to calculate the correlate parameters of oridonin to different genera plasma proteins.
Methods: The binding rates of oridonin with different genera plasma proteins were determined by equilibrium dialysis method. The concentrations of oridonin were assayed by HPLC.
Results: The binding rates of oridonin with rat, human plasma and BSA 69.66±12.8%, 59.62±12.6%, 57.94±4.1%, 78.15±3.6%, 77.92±8.8%, 76.72±7.3%, 35.58±7.2%, 34.59±10.8%, 32.03±6.0%, respectively.
Conclusion: The equilibrium dialysis method was applied to study the the protein binding rates of oridonin. The binding rates of oridonin to human plasma protein, rat plasma protein and BSA were in middle level. They were a kind of middle 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 oridonin is not dependent on the doses.It is suggested that the above results may ensure to have safety of oridonin in clinic.
Part seven Studies on absorption kinetics of oridonin in intestines in rats
Objective: To develop a high-performance liquid chromatography coupled with diode array detection (HPLC/DAD) method for simultaneous determination of oridonin and phenolsulfonphthalein in the circulation solution and to investigate the absorption kinetics of oridonin 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. Oridonin concentration in the flux was measured by the reversed phase HPLC. The chromatographic procedure was carried out with Diamonsil C18 (250 mm×4.6mm, 5μm) as an analytic column and a mixture consisting of methanol- acetonitrile-0. 5% phosphoric acid(55:15:30)as mobile phase at the temperature of column of 30?C. The detection wavelength was set at 238 nm for oridonin and phenolsulfonphthalein and the flow rate was 1.0mL·min~(-1).
Results: When the concentration was raised from 5.0 to 15.0μg·mL~(-1), the uptake of oridonin was increased linearly. Concentration had no effect on the permeability coefficient. The permeability coefficients of oridonin at duodenum, jejunum, ileum and colon were ( 0.0475±0.0062 ),(0.0468±0.0051),(0.0346±0.0037),(0.0435±0.0023)h~(-1), respectively.
Conclusion: It is the first time to use sensitive, accurate, and simple HPLC/DAD method to determine oridonin and phenolsulfonphthalein in the circulation solution simultaneously. The absorption of oridonin in rat’s intestine is a first-order process with the passive diffusion mechanism. Oridonin can be absorbed in whole intestinal segments. So oridonin sustained-released formulations can be prepared.
引文
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