High-performance liquid chromatographic determination and metabolic study of sennoside a in daiokanzoto by mouse intestinal bacteria

Daiokanzoto (DKT, combination of rhubarb and glycyrrhiza), a Kampo medicine, is clinically effective for constipation. Sennoside A is well known to induce diarrhea. Sennoside A is a prodrug that is transformed into an active metabolite, rheinanthrone, by intestinal bacteria. In this study, we investigated the effects of glycyrrhiza on the activity of sennoside A metabolism in intestinal bacteria using mouse feces. A high-performance liquid chromatography (HPLC) method for the determination of sennoside A in incubation mixture of DKT with mouse feces was established. The retention time of sennoside A was 9.26±0.02 min with a TSKgel ODS-80TsQA column by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile and detection at 265 nm. We found that the activity of sennoside A metabolism in intestinal bacteria was significantly accelerated when glycyrrhiza, liquiritin or liquiritin apioside coexisted with sennoside A, whereas that of glycyrrhizin was not altered. This method is applicable for determination of the activity of sennoside A metabolism by anaerobic incubation of DKT with mouse feces.     

Determination of Paeoniflorin in Total Glucosides of Peony Root by HPLC

The crude drug of Radix Paeoniae Alba has been used extensively in traditional Chinese medicine as an analgesic, an antispasmodic,an astringent, and a sedative for the treatment of a variety of painful afflictions. A modern way of using Radix Paeoniae Alba is the application of Total Glucosides of Peony Root (TGPR), which is the ethanol extract from the crude drug. Our interest in this work is to develop a method to determine one of the main effective components, Paeoniflorin, in TGPR by HPLC. Although several HPLC methods for the determination of Paeoniflorin were reported before,our method has the advantages of simplicity, rapidity and accuracy.     

液质联用技术研究人参与干姜或赤芍配伍前后人参皂苷及其抗氧化活性的变化

采用高效液相色谱与电喷雾质谱联用技术(HPLC-ESI-MS),对不同质量比的人参与干姜或赤芍配伍过程中人参皂苷的变化进行了研究,发现随加入的干姜量增加,共煎液中的人参皂苷含量依次降低;少量的赤芍可以使各皂苷的溶出量增加;同时测定了人参单煎液、人参与干姜、赤芍共煎液中正丁醇提取物和水提物的抗氧化活性。 以抗坏血酸(500 μmol/L)作对照,人参与干姜、赤芍配伍溶液的抗氧化活性比人参单煎液要好,同时人参与干姜、赤芍共煎液中正丁醇提取物的FRAP(铁离子还原/抗氧化能力测定)值分别为1562.29和2969.78 μmol/L,高于人参与2种药单煎液（1260.27和2502.07 μmol/L）之和。     

Preparative Chromatographic Isolation of Caulophine from Radix caulophylli and its Metabolism In Vivo and In Vitro

Caulophine, as a novel alkaloid, was found in Radix caulophylli and has anti-myocardial ischemia activities. To conduct product development research on Radix caulophylli and caulophine, a preparative high-performance liquid chromatography method for the preparation of caulophine was investigated. Preparative HPLC was optimized to allow fraction I to be separated first and the caulophine was isolated following the second round of preparative HPLC. The purity of caulophine was >98%, which was assessed using analytical HPLC. Then, a highly sensitive and specific liquid chromatography-mass spectrometric method for the excretion and metabolism of caulophine in vivo was investigated. The metabolism to caulophine glucuronide conjugate was studied in rat liver microsomes or dog liver microsomes in vitro systems. Biosamples were pretreated by solid phase extraction (SPE) and analyzed by LC-MS with electrospray ionization (ESI) interface. Method validation results showed within-day and between-day precision was 1.14–6.21% and 5.45–9.78%, respectively, and average recoveries for all matrices were greater than 80%. The limits of detection for this method were determined to be up to 1 ng · mL^?1 of caulophine. Excretion of caulophine in rat results indicated that the total cumulative excretion of caulophine was high, with greater than 50% of the treatment dose being excreted. Two metabolites including glucuronide conjugate and N-oxide of caulophine were found in rat urine and feces by LC-MS. Moreover, the same caulophine glucuronide conjugate was observed in rat liver microsomes system.     

Study on the nitric oxide generating effects from aristolochic acid in vitro

In this study, an in vitro nitric oxide (NO)-assay system based on the Griess reaction was used to investigate the (NO)-generating effects of aristolochic acid (AA) for the first time. AA was separated into its different components, aristolochic acid I (AAI) and aristolochic acid II (AAII), by preparative HPLC. AAI and AAII were incubated with human intestine bacteria (HIB) or rat intestine bacteria (RIB). A NO mixture generated from AAI and AAII by intestinal bacteria was observed and denitroso metabolites of AAI or AAII were detected in vitro by liquid chromatography/tandem mass spectrometry. Therefore, NO generation might be closely related to the metabolic process of AA in vitro. It suggested that one possible mechanism for the toxicity of AA may be due to the generation of NO from these compounds by intestinal bacteria.     

Kinetic distribution of paeoniflorin in cortex of normal and cerebral ischemia-reperfusion rats after intravenous administration of Paeoniae Radix extract

The time course of paeoniflorin in the cortex of normal and cerebral ischemia-reperfusion rats, following intravenous administration of Paeoniae Radix extract at a dose of 60 mg/kg of paeoniflorin, was determined using high-performance liquid chromatographic (HPLC) assay. The results showed that paeoniflorin could penetrate through the blood-brain barrier to reach the cortex, and that the injuries of ischemia-reperfusion could play an important role in pharmacokinetic process of paeoniflorin in the cortex after intravenous administration of Paeoniae Radix extract. The cortex concentrations of paeoniflorin in cerebral ischemia-reperfusion rats were lower 5 min after dosing and declined more slowly than that in normal control.     

益气通痹胶囊质量标准研究

采用薄层色谱法对益气通痹胶囊中黄芪、何首乌、五味子、赤芍、延胡索进行定性鉴别,采用高效液相色谱法测定了制剂中淫羊藿苷的含量. 所用薄层色谱具有鉴别特征,色谱斑点清晰,专属性强. 淫羊藿苷在0.55~3.30 μg范围内呈良好的线性关系(r =0.99996),平均回收率为98.4%,RSD为0.62 %. 所建立的定性和定量方法,操作简便可靠,专属性强,重现性好,能较全面反映该制剂内在质量,可作为益气通痹胶囊新药研发质量控制标准.     

Static headspace‐multicapillary column with gas chromatography coupled to ion mobility spectrometry as a simple approach for the discrimination of crude and processed traditional Chinese medicines

The processing procedure can alter the nature and chemical transformation of traditional Chinese medicine to accommodate different clinical dispensing and preparation requirements. In this study, static headspace‐multicapillary column with gas chromatography coupled to ion mobility spectrometry was developed for the rapid and sensitive discrimination of crude and processed traditional Chinese medicine. Using Radix Paeoniae Alba as a traditional Chinese medicine model, the combined power of this approach was illustrated by classifying the crude and processed Radix Paeoniae Alba samples into two main categories. The contents of the main components in Radix Paeoniae Alba varied significantly. The established method could promote the use of ion mobility spectrometry in intrinsic quality control and differentiation of herbal medicines from other processed products or preparations.     

LC-MS/MS determination and pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang-Min-Ling-Wan

A rapid, sensitive and selective liquid chromatography/tandem mass spectrometry method (LC‐MS/MS) was developed and validated for simultaneous determination of albiflorin and paeoniflorin in rat plasma using geniposide as an internal standard. Plasma samples were extracted by solid‐phase extraction. Chromatographic separation was carried out on a Zorbax SB‐C₁₈ analytical column (150 × 2.1 mm × 5 µm) with 0.1% formic acid–acetonitrile (70:30, v/v) as the mobile phase. Detection was performed by multiple reaction monitoring mode using electrospray ionization in the positive ion mode. The total run time was 3.0 min between injections. The calibration curves were linear over a range of 1–1000 ng/mL for albiflorin and 2–2000 ng/mL for paeoniflorin. The overall precision and accuracy for all concentrations of quality controls and standards were better than 15%. Mean recovery was determined to be 87.7% for albiflorin and 88.8% for paeoniflorin. The validated method was successfully applied to the pharmacokinetic study of albiflorin and paeoniflorin in rat plasma after oral administration of Radix Paeoniae Alba extract and Tang‐Min‐Ling‐Wan. The pharmacokinetic parameters showed that albiflorin and paeoniflorin from Tang‐Min‐Ling‐Wan were absorbed more rapidly with higher concentrations in plasma than that from Radix Paeoniae Alba extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine. Copyright © 2010 John Wiley & Sons, Ltd.     

Species differentiation and quality assessment of Radix Paeoniae Rubra (Chi-shao) by means of high-performance liquid chromatographic fingerprint

There are two species under the monograph of Radix Paeoniae Rubrae ("Chi-shao" in Chinese) in Chinese Pharmacopoeia 2005 edition-Paeonia lactiflora Pallas and Paeonia veitchii Lynch. Due to different species and growing conditions, there are significant chemical differences between the two species, which may result in the improper clinical usage under the same name. Chemical pattern expressed by high performance liquid chromatographic (HPLC) fingerprint analysis can play an important role in species differentiation and quality control of Radix Paeoniae Rubra. In the present work, HPLC fingerprints of two kinds of Radix Paeoniae Rubra have been established and analysed with chemometric methods including similarity evaluation and principal component analysis. Both of the fingerprint common patterns of the two species comprise 13 characteristic peaks, nine of which were common peaks of the two species. However, significant differences between the roots of P. veitchii and P. lactiflora exist not only in the content of certain constituents, especially phenolic acids but also in peak-to-peak ratios expressed by the fingerprint patterns. According to the recent pharmacological studies on polyphenolic constituents, root originating from P. veitchii may possess better efficacy and quality than that from P. lactiflora. Our research reveals that further pharmacological investigation is very necessary to determine whether the two species should be embodied under the same monograph in Chinese Pharmacopoeia.     

利用液质联用技术分析干姜对乌头类生物碱在大鼠肠内吸收的影响

为从吸收的角度考察干姜对乌头类双酯型生物碱的解毒机理, 采用外翻肠囊法展开实验. 利用超高液相与三重四极杆质谱联用技术定量检测双酯型生物碱成分, 采用标准曲线法计算乌头碱、中乌头碱、次乌头碱在肠囊内吸收的绝对含量, 采用质谱峰面积直接分析其它双酯型生物碱的相对变化, 结果加入干姜提取液后, 乌头碱、中乌头碱、次乌头碱的单位肠管面积累计吸收量均降低, 10-羟基中乌头碱的的累积峰面积降低; 加入维拉帕米后, 双酯型生物碱的单位肠管面积累计吸收量及累积峰面积均增加; 向含有地高辛的肠营养液中加入干姜提取液后, 地高辛在各实验时间点的单位肠管面积累计吸收量均降低, 根据以上结果推测干姜抑制乌头类双酯型生物碱在大鼠肠囊内吸收的可能机制是通过诱导肠内P-葡糖蛋白, 从而抑制作为P-葡糖蛋白底物的双酯型生物碱的吸收, 最终起到减毒作用.     

High performance liquid chromatography-mass spectrometry analysis for rat metabolism and pharmacokinetic studies of lithospermic acid B from danshen

Metabolism and pharmacokinetic studies on rat were conducted for lithospermic acid B, one of the components from Radix Salviae Miltiorrhizae (danshen) that shows many bioactivities. Liquid chromatography-electrospray ionization mass spectrometry method was applied for the determination of lithospermic acid B and its metabolites in samples from in vitro and in vivo metabolism studies. Rat plasma samples collected after intravenous administration were analyzed for obtaining pharmacokinetic data of lithospermic acid B. Four O-methylated metabolites, namely one monomethyl-, two dimethyl- and one trimethyl-lithospermic acid B, were detected when lithospermic acid B was incubated in rat hepatic cytosol. These four metabolites were also detected in rat bile, plasma and feces samples after intravenous administration of lithospermic acid B. The in vitro and in vivo results indicate that the methylation is the main metabolic pathway of lithospermic acid B. The danshen component and its methylated metabolites were excreted to rat bile and feces.     

Determination of paeoniflorin, ferulic acid and baicalin in the traditional Chinese medicinal preparation Dang-Guei-San by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of paeoniflorin, ferulic acid and baicalin in the traditional Chinese medicinal preparation Dang-Guei-San, which contains Paeoniae Radix, Swertiae Herba, Cnidii Rhizoma and Scutellariae Radix, was established. The samples were separated with a Cosmosil 5C₁₈-AR column by gradient elution with 0.03% (v/v) phosphoric acid- acetonitrile (0 min 96:4, 5 min 84:16, 7 min 82:18, 14–30 min 78:22) as the mobile phase at a flow-rate of 1.0 ml/min, with detection at 245 nm. Methylparaben was used as the internal standard and three equations were derived showing linear relationships between the peak- area ratios of marker components (paeoniflorin, ferulic acid and baicalin) to methylparaben and concentration. The recoveries of paeoniflorin, ferulic acid and baicalin were 27.86, 33.89 and 49.31%, respectively. The repeatability (relative standard deviation) was generally less than 5% (n = 5). The effects of various processes such as concentration by reduced-pressure evaporation, freeze-drying and spray-drying were studied and commercial concentrated herbal preparations containing Paeoniae Radix, Swertiae Herba, Cnidii Rhizoma and Scutellariae Radix were also analysed.     

Effect of pH on the Metabolism of Aconitine under Rat Intestinal Bacteria and Analysis of Metabolites Using HPLC/MS-MSn Technique

A semi‐quantitative method of mass spectrometry (MS) has been described for the analysis of metabolites of aconitine by rat intestinal bacteria at different pH. At pH 7.0, the rat intestinal bacteria exhibit optimal activity for the metabolism of aconitine. A high‐performance liquid chromatography‐electrospray ionization multiple‐stage mass spectrometry (HPLC/ESI‐MSⁿ) method has been applied to investigate the characteristic product ions of metabolites. Then, the logical fragmentation pathways of metabolites have been proposed. By comparing the retention time (t_R) of HPLC and the ESI‐MSⁿ data with the data of standard compounds and reports from literature, ten metabolites have been identified and a distinctive metabolite (15‐deoxyaconitine) has been deduced first time. The experimental results demonstrate that HPLC/ESI‐MSⁿ is a specific and useful method for the identification of metabolites of aconitine. Also, in the present paper, the HPLC‐MS method was introduced to determine the synthetical metabolite prior to the study of the toxicity by the method of Bliss.     

Interaction of the main components from the traditional Chinese drug pair Chaihu-Shaoyao based on rat intestinal absorption

The Chaihu-Shaoyao drug pair (Bupleuri Radix and Paeoniae Radix Alba) which is a traditional Chinese drug pair, has been widely used for anti-inflammatory purposes. Saikosaponin a (SSA), saikosaponin d (SSD) and paeoniflorin are identified as the main components in the pair. The present study focused on the interaction of the main components based on investigating their intestinal absorption using a four-site perfused rat intestinal model in order to clarify the mechanism of the compatibility of Chaihu-Shaoyao. The concentrations of SSA, SSD and paeoniflorin in the intestinal perfusate were determined by LC/MS or UPLC (Ultra Performance Liquid Chromatography) methods, followed by P*(eff) (effective permeability) and 10% ABS (the percent absorption of 10 cm of intestine) calculations. The results showed that all of the three main components displayed very low permeabilities (P*(eff) < 0.4), which implied their poor absorption in the rat intestine. The absorption levels of SSA and SSD were similar in intestine and higher in ileum than those in other intestinal regions in the decreasing order: colon, jejunum and duodenum. However, there is no significant difference in the absorption of paeoniflorin in the four segments (P < 0.05). The P*(eff) values of paeoniflorin exhibited an almost 2.11-fold or 1.90-fold increase in ileum when it was co-administrated with SSA and SSD, as well as 2.42-, 2.18-fold increase in colon, respectively, whereas the absorptions of SSA and SSD were not influenced by paeoniflorin. In conclusion, SSA and SSD could promote the absorption of paeoniflorin. To some extent this might explain the nature of the compatibility mechanisms of composite formulae in TCMs.     

Determination of renin activity in human plasma by column-switching high-performance liquid chromatography with fluorescence detection

A column-switching high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the determination of the renin activity in human plasma. The method is based on the quantification of the enzymatically produced angiotensin I. Angiotensin I liberated from a synthetic substrate (tridecapeptide of human angiotensinogen) and [Val5]-angiotensin I as an internal standard are converted into fluorescent derivatives by reaction with benzoin. The derivatives are separated from various interfering substances by column-switching HPLC using three reversed-phase columns. The limit of detection (signal-to-noise ratio = 3) of the renin activity is 2.7 pmol of angiotensin I formed per h per ml of plasma, which corresponds to approximately 820 fmol of angiotensin I injected. The column-switching method in combination with pre-column derivatization for the fluorimetric detection permits the sensitive and selective determination of the enzymatically formed angiotensin I. Hence low activities of renin in normal human plasma are readily measured.     

Microbial metabolism of loganin by intestinal bacteria and identification of new metabolites in rat

Loganin is an important constituent of the traditional Chinese medicine Fructus Corni, with several bioactivities. Microbial metabolism of loganin by intestinal bacteria was investigated. Two metabolites (log-1 and log-2) were isolated from anaerobic culture and their structures were identified by means of their ESI-MS, (1)H-NMR, (13)C-NMR and 2D-NMR spectral data. Log-1 was an aglycone of loganin and log-2 was proved to be a new compound. In vivo metabolites of loganin were detected in rat urine, bile and feces after oral administration of loganin and the structures were proved to be identical with that of the microbial metabolites log-1 and log-2 by HPLC-PDA analysis and comparison with the reference standards. Therefore we can prepare metabolites by anaerobic culture with intestinal bacteria.     

Simultaneous determination of indole metabolites of tryptophan in rat feces by chemical labeling assisted liquid chromatography-tandem mass spectrometry

As the connecting part of diet and host physiology, intestinal microbes can convert the ingested diet into a huge number of physiologically active small molecules. Indole metabolites of tryptophan are precursors or signal molecules for many biologically active substances, which are involved in serotonin and microbial catabolism pathways. To understand the influence of tryptophan metabolism in the intestinal environment on the neurological and immune systems at the molecular level, it is important to establish a high-coverage analytical method to comprehensively analyze the metabolites involved in tryptophan metabolism. However, due to a small molecular weight and poor response during mass spectrometry analysis, as well as weak retention on the reversed-phase chromatography, determination of indole metabolites of tryptophan is challenging. Here, we proposed a method for the simultaneous determination of 20 indole metabolites of tryptophan in a single run on reversed-phase chromatography by chemical labeling coupled to liquid chromatography-tandem mass spectrometry analysis. 4-(Dimethylamino)benzaldehyde (DMAB) was used for the labeling of indole metabolites of tryptophan, which could significantly improve the detection sensitivities and retention of these metabolites on reversed-phase chromatography. With the developed method, we realized the sensitive detection and comprehensive analysis of 15 endogenous indole metabolites of tryptophan in rat feces samples with functional dyspepsia intervention by acupuncture. The developed method offers a useful tool for studying tryptophan metabolism-related diseases.     

色谱指纹谱用于中药大黄抗肿瘤活性成分的筛选

采用液相色谱-质谱联用方法分析了中药大黄经过SD大鼠肝匀浆体外代谢前后的指纹谱中色谱峰面积、保留值的差异。指出5种游离型蒽醌化合物在SD大鼠肝匀浆体外代谢体系中只有大黄酚发生代谢反应转化为芦荟大黄素。考察了体外代谢条件下,肝匀浆浓度与代谢时间对大黄酚转化及其代谢产物的影响。SD大鼠体外抗肿瘤试验表明,大黄代谢物对于人宫颈癌(HeLa)细胞的抑制活性略高于其提取物。通过比较芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚的活性,并结合大黄酚的体外代谢反应的考察,解释了大黄代谢物对肿瘤细胞活性的抑制率的提高是由大黄酚的代谢产物芦荟大黄素浓度的增加引起的。