Simultaneous quantitation of 14 active components in Yinchenhao decoction by using ultra high performance liquid chromatography with diode array detection: Method development and ingredient analysis of different commonly prepared samples

We developed a novel quantitative analysis method based on ultra high performance liquid chromatography coupled with diode array detection for the simultaneous determination of the 14 main active components in Yinchenhao decoction. All components were separated on an Agilent SB‐C18 column by using a gradient solvent system of acetonitrile/0.1% phosphoric acid solution at a flow rate of 0.4 mL/min for 35 min. Subsequently, linearity, precision, repeatability, and accuracy tests were implemented to validate the method. Furthermore, the method has been applied for compositional difference analysis of 14 components in eight normal‐extraction Yinchenhao decoction samples, accompanied by hierarchical clustering analysis and similarity analysis. The result that all samples were divided into three groups based on different contents of components demonstrated that extraction methods of decocting, refluxing, ultrasonication and extraction solvents of water or ethanol affected component differentiation, and should be related to its clinical applications. The results also indicated that the sample prepared by patients in the family by using water extraction employing a casserole was almost same to that prepared using a stainless‐steel kettle, which is mostly used in pharmaceutical factories. This research would help patients to select the best and most convenient method for preparing Yinchenhao decoction.     

四君子汤和四物汤微量元素含量研究

通过了解中药“四君子汤”和“四物汤”中微量元素含量的异同,研讨了微量元素和中医“气血”理论之间的关系。实验表明,“四君子汤”和“四物汤”在微量元素的含量方面,有一定的类同和差异。该现象同中医的“气血”理论有非常相像的地方。同时发现中药中的微量元素在煎煮过程中,并不是同想象的一样匀速进入药液,而是不同的元素在不同的时间段不同的批次进入药液,这也提示中药煎法理论有一定依据。由于目前做的实验还少,本实验留下很多问题和疑点,值得进一步做有关的实验和研究。     

阴离子交换色谱-积分脉冲安培检测法测定寿胎丸中游离氨基酸

建立了阴离子交换色谱-积分脉冲安培检测法测定寿胎丸煎煮液中游离氨基酸的分析方法。实验考察了18种氨基酸的梯度洗脱条件,优化了固相萃取条件,最终选用SSDBX型固相萃取小柱净化煎煮液。在优化实验条件下,进行了加标回收实验,回收率在71.2%~120.9%之间,相对标准偏差为0.8%~10.4%。18种氨基酸的检测限为0.07~5.00μg/L。方法能满足寿胎丸煎煮液中氨基酸的检测要求。     

固定化脂质体色谱研究中药复方的细胞膜通透性成分及其质量控制

提出了固定化脂质体色谱(immobilized liposome chromatography,ILC)研究中药复方的细胞膜通透性成分及其质量控制的方法。以当归补血汤及其组分为例,考察了它们在ILC色谱柱上的分离效果;当归补血汤水提取液和75％乙醇提取液在ILC色谱柱上各有8个保留峰,当归水提取液、甲醇提取液和75％乙醇提取液在ILC色谱柱上分别有3、6、9个保留峰,黄芪水提取液、甲醇提取液和75％乙醇提取液在ILC色谱柱上各有7个保留峰;建立了当归补血汤及其组分中阿魏酸和蒿本内酯的定量分析方法,当归补血汤水提取液和75％乙醇提取液中阿魏酸的含量分别为0．0743％和0．0688％,蒿本内酯的含量分别为0．0472％和0．457％,当归水提取液和75％乙醇提取液中阿魏酸的含量分别为0．0694％和0．0691％,蒿本内酯的含量分别为0．0781％和0．455％。     

Simultaneous determination of naringenin and hesperetin in rats after oral administration of Da-Cheng-Qi decoction by high-performance liquid chromatography-tandem mass spectrometry

To quantify naringenin and hesperetin in rat plasma after oral administration of Da-Cheng-Qi decoction, a famous purgative traditional Chinese medicine, a high-performance liquid chromatography-tandem mass spectrometry method was developed and validated. The HPLC separation was carried out on a Zorbax SB-C(18) column using 0.1% formic acid-methanol as mobile phase and estazolam as internal standard after the sample of rat plasma had been cleaned up with one-step protein precipitation using methanol. Atmospheric pressure chemical ionization in the positive ion mode and selected reaction monitoring method was developed to determine the active components. This method was validated in terms of recovery, linearity, accuracy and precision (intra- and inter-batch variation). The recoveries of naringenin and hesperetin were 72.8-76.6 and 75.7-77.2%, respectively. Linearity in rat plasma was observed over the range of 0.5-250 ng/mL (r2 > 0.99) for both naringenin and hesperetin. The accuracy and precision were well within the acceptable range and the relative standard deviation of the measured rat plasma samples was less than 15% (n = 5). The validated method was successfully applied for the evaluation of the pharmacokinetics of naringenin and hesperetin administered to six rats.     

Identification of metabolites in WZS‐miniature pig urine after oral administration of Danshen decoction by HPLC coupled with diode array detection with electrospray ionization tandem ion trap and time‐of‐flight mass spectrometry

Danshen (DS) is a widely used traditional Chinese medicine for treating cardiovascular and cerebrovascular diseases. A simple, rapid and sensitive method was developed for identification of the in vivo metabolites in urine of WZS‐miniature pigs after oral administration of DS decoction by HPLC coupled with diode array detection with electrospray ionization tandem ion trap and time‐of‐flight mass spectrometry. This method has been successfully applied to simultaneous identification of 50 compounds (including 11 new ones) in pig urine. In addition, one new compound, (3‐hydroxyphenyl) crylic acid glycine methyl ester (C1), along with eight known ones were first isolated by column chromatography and identified by spectroscopic means, including 1D/2DNMR and mass spectrometry, as reference substances. Ten phenolic compounds (protocatechuic aldehyde, protocatechuic acid, caffeic acid, danshensu, ferulic acid, isoferulic acid, rosmarinic acid and salvianolic acid A/B/D) were found to be the main absorbed original constituents of DS decoction, which underwent the metabolic reactions of glucuronidation, sulfation, methylation, hydrogenation and glycine conjugation in vivo. In conclusion, the developed method is applicable to the analysis and identification of constituents in biological matrices after administration of DS decoction. Copyright © 2012 John Wiley & Sons, Ltd.     

HPLC method for the determination and pharmacokinetic studies on geniposide in rat serum after oral administration of traditional Chinese medicinal preparation Yin-Zhi-Ku decoction

A new HPLC method for the determination of geniposide in rat serum with solid-phase extraction (SPE) for preconcentration is described. Geniposide and an internal standard (paeoniflorin) were extracted from serum by SPE using C18 cartridges. Analysis of the extract was then performed on a reversed-phase C18 column using acetonitrile-water (16:84, v/v) as the eluting solvent system, and UV detection at 238 nm was used to measure the analyte with a limit of quantitation about 0.1 microg/mL. The calibration curve for geniposide was linear (r = 0.9993) in the concentration range 0.1-16.0 microg/mL. The intra- and inter-day precision of the geniposide were determined and their RSD did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of geniposide from rat serum after oral administration of Yin-Zhi-Ku decoction.     

白术煎剂表面增强拉曼光谱分析

测试分析了白术煎剂及其在银胶中的拉曼光谱,并对其进行初步谱峰归属。白术煎剂表面增强拉曼光谱在396,548,617,730,955和1 327 cm-1处,出现6个明显的拉曼信号。测试了白术煎剂和白术煎剂-银胶混合体系的紫外-可见吸收光谱,该吸收光谱在长波区出现新共振吸收峰(999 nm处),进而研究了白术煎剂在银溶胶上的吸附特性及其表面增强机理。结果表明,表面增强拉曼光谱可能为白术煎剂或其他中药煎剂提供一种准确、直接、快速的检测方法。     

补阳还五汤联合依达拉奉对脑缺血再灌注损伤后神经细胞凋亡的影响

目的研究补阳还五汤联合依达拉奉对小鼠脑缺血再灌注损伤后神经细胞凋亡及Bcl-2、Bax和Caspase-3表达的影响。方法将50只小鼠随机分为假手术组、模型组、补阳还五汤组、依达拉奉组和两药联用组,每组10只,其中缺血再灌注后1d和7d各5只。首先制备小鼠大脑中动脉缺血模型,TUNEL法观察神经细胞凋亡指数（AI）,免疫组化法观察小鼠大脑皮质神经元Bcl、Bax-2和Caspase-3表达阳性细胞数。结果与模型组比较,补阳还五汤组、依达拉奉组、两药联用组小鼠脑神经细胞AI值均明显降低（P＜0.05）,Bcl-2/Bax比值明显升高（P＜0.05）,Caspase-3表达阳性细胞数明显减少（P＜0.05）,其中又以两药联用组更为明显（P＜0.05）。结论两药联用能降低脑缺血再灌注损伤后神经细胞的凋亡,降低促凋亡基因Caspase-3的表达,协同发挥脑保护作用。