Journal of Chromatography B (v.1026, #C)

Direct analysis of traditional Chinese medicines by mass spectrometry by Melody Yee-Man Wong; Pui-Kin So; Zhong-Ping Yao (2-14).
Analysis of traditional Chinese medicines (TCMs) plays important roles in quality control of TCMs and understanding their pharmacological effects. Mass spectrometry (MS) is a technique of choice for analysis of TCMs due to its superiority in speed, sensitivity and specificity. However, conventional MS analysis of TCMs typically requires extensive sample pretreatment and chromatographic separation, which could be time-consuming and laborious, prior to the analysis. The expanding usage of TCMs worldwide demands development of rapid, cost-effective and reliable methods for analysis of TCMs. In recent years, new sample preparation and ionization techniques have been developed to enable direct analysis of TCMs by MS, significantly reducing the analysis time and cost. In this review, various MS-based techniques, mainly including ambient ionization-MS and MALDI-MS based techniques, applied for direct analysis of TCMs are summarized and their applicability and future prospects are discussed.
Keywords: Traditional Chinese medicines; Mass spectrometry; Direct analysis; Ambient ionization; Matrix-assisted laser desorption/ionization;

Traditional Chinese medicines (TCMs) are gaining more and more attentions all over the world. The focus of TCMs researches were gradually shifted from chemical research to the combination study of chemical and life sciences. However, obtaining precise information of TCMs process in vivo or in vitro is still a bottleneck in bioanalysis of TCMs for their chemical composition complexity. This paper reviewed the recent analytical methods especially mass spectrometry technology in the bioanalysis of TCMs, and data processing techniques in the qualitative and quantitative analyses of metabolite of TCMs. Additionally, the difficulties encountered in the analyzing biological samples in TCMs and the solutions to these problems have been mentioned.
Keywords: Traditional Chinese medicines; Mass spectrometry; Bioanalysis; Metabolites;

Current application of chemometrics in traditional Chinese herbal medicine research by Yipeng Huang; Zhenwei Wu; Rihui Su; Guihua Ruan; Fuyou Du; Gongke Li (27-35).
Traditional Chinese herbal medicines (TCHMs) are promising approach for the treatment of various diseases which have attracted increasing attention all over the world. Chemometrics in quality control of TCHMs are great useful tools that harnessing mathematics, statistics and other methods to acquire information maximally from the data obtained from various analytical approaches. This feature article focuses on the recent studies which evaluating the pharmacological efficacy and quality of TCHMs by determining, identifying and discriminating the bioactive or marker components in different samples with the help of chemometric techniques. In this work, the application of chemometric techniques in the classification of TCHMs based on their efficacy and usage was introduced. The recent advances of chemometrics applied in the chemical analysis of TCHMs were reviewed in detail.
Keywords: Traditional Chinese herbal medicine; Chemometrics; Quality control; Pharmacological efficacy; Bioactive components;

UPLC-Q-TOF-MSE analysis of the constituents of Ding-Zhi-Xiao-Wan, a traditional Chinese antidepressant, in normal and depressive rats by Lu Xu; Li-Hua Mu; Jie Peng; Wan-Wan Liu; Xiao Tan; Zhao-Liang Li; Dong-Xiao Wang; Ping Liu (36-42).
Ding-Zhi-Xiao-Wan (DZXW) is a traditional Chinese medicine widely used for treating depression. To clarify the bioactive constituents of DZXW, a new rapid ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MSE) method was established in this study, with the whole extract of the formula separated into multiple components to facilitate the analytical process. In total, 97 compounds were detected and 88 were identified in DZXW. Based on their exact masses, fragmentation characteristics, and retention times, 85 of the 88 compounds were confirmed either conclusively or tentatively, and three potentially novel compounds were identified. In addition, following a three-day oral administration of DZXW, 60 and 28 compounds were observed in the plasma of normal and depressive rats, respectively. Finally, by combining our data with pharmacological information, 10 compounds were predicted as the likely bioactive constituents of DZXW as an antidepressant agent. Our approach provided a rapid method for characterising the chemical constituents of DZXW, and we were the first to screen for bioactive indexes in the plasma of depressive rats. Furthermore, our results provide useful chemical information that could be employed for further study of the pharmacodynamic material basis of DZXW's antidepressant effects.
Keywords: Ding-Zhi-Xiao-Wan; UPLC-Q-TOF-MS; Chemical constituents; Metabolites; CUMS; Depression;

Characterization of chemical constituents and rats metabolites of an alkaloidal extract of Alstonia scholaris leaves by liquid chromatography coupled with mass spectrometry by Jing Cao; Hong-Mei Shen; Qi Wang; Yi Qian; Hong-Cheng Guo; Kai Li; Xue Qiao; De-An Guo; Xiao-Dong Luo; Min Ye (43-55).
Alstonia scholaris has been used in “Dai” ethnic medicine to treat chronic respiratory diseases for a long history, and the major bioactive constituents are alkaloids. An alkaloidal extract of A. scholaris leaves (AAS) has been developed into an investigational new drug, and has been approved for phase I/II clinical trials by China Food and Drug Administration. However, little is known on the chemical composition and in vivo metabolism of AAS, thus far. In this study, an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC/qTOF-MS) method was established to characterize the chemical constituents of AAS. Samples were separated on an ACQUITY UPLC CSH column (2.1 × 100 mm, 1.7 μm) with acetonitrile and water containing 0.3% formic acid as the mobile phase. On the basis of high-accuracy mass spectral analysis, a total of 35 alkaloids were characterized from AAS, including 11 scholaricine-type, 9 vallesamine-type, 12 picrinine-type, and 3 tubotaiwine-type alkaloids. Furthermore, the metabolic pathways of 4 representative alkaloids in rats were studied. They mainly undertook hydroxylation and glucuronidation reactions. Based on the above metabolic pathways, the metabolism of AAS (10 mg/kg) in rats after oral administration was studied by LC/MS. A total of 33 compounds in plasma, 40 compounds in urine, and 38 compounds in feces were characterized. The results indicated that scholaricine-type alkaloids could get into circulation more readily than the other types. This is the first systematic study on chemical profiling and metabolites identification of AAS.
Keywords: Alstonia scholaris; Alkaloids; UHPLC/qTOF-MS; Metabolites identification; Herbal medicine;

Bioactive heterocyclic alkaloids with diterpene structure isolated from traditional Chinese medicines by Tengfei Xu; Shu Liu; Lulu Meng; Zifeng Pi; Fengrui Song; Zhiqiang Liu (56-66).
The diterpenoid alkaloids as one type of heterocyclic alkaloids have been found in many traditional herbal medicines, such as genus Consolida, Aconitum, and Delphinium (Ranunculaceae). Pharmacological researches have indicated that many diterpenoid alkaloids are the main bioactive components which have analgesic, anti-inflammatory, anti-microbial, anti-tumor, cardiotonic, and anti-arrhythmic activities. Studies focused on the determination, quantitation and pharmacological properties of these alkaloids have dramatically increased during the past few years. Up to now, newly discovered diterpenoid alkaloids with important biological activities have been isolated and synthesized. Considering their significant role and diffusely used in many disease treatments, we summarized the information of their analysis methods, bioactivity, metabolism and biotransformation in vivo as well as the pharmacological mechanisms. Based on above review, the further researches are suggested.
Keywords: Diterpendoid alkaloids; Analysis methods; Biological activity; Metabolism; Biotransformation;

Separation and characterization of bufadienolides in toad skin using two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography coupled with mass spectrometry by Yun Zhang; Hongli Jin; Xiaolong Li; Jianqiang Zhao; Xiujie Guo; Jixia Wang; Zhimou Guo; Xiuli Zhang; Yanduo Tao; Yanfang Liu; Deliang Chen; Xinmiao Liang (67-74).
Bufadienolides possess various bioactivities especially antitumor. Due to the high structural diversity, the separation of bufadienolides often suffers from coelution problem on conventional RP columns. In this work, an off-line two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography (2D-NPLC × RPLC) method was developed to separate and characterize bufadienolides in toad skin. Several RP and NP columns were evaluated with five reference bufadienlides. The XUnion C18 and XAmide columns exhibited superior chromatographic performances for bufadienlide separation, and were selected in RPLC and NPLC, respectively. RPLC was used in the second-dimension for the good compatibility with MS, while NPLC was adopted in the first-dimension. The orthogonality of the 2D-NPLC × RPLC system was investigated by the geometric approach using fifteen bufadienolide mixtures. The result was 49.6%, demonstrating reasonable orthogonality of this 2D-LC system. By combining the 2D-LC system with MS, 64 bufadienlides including 33 minor ones and 11 pairs of isomers in toad skin were identified. This off-line 2D-NPLC × RPLC allowed to solve the coelution problem of bufadienlides in one-dimension RPLC, and thus facilitated the identification significantly.
Keywords: Bufadienolides; Normal-phase liquid chromatography; Two-dimensional liquid chromatography; MS/MS; Toad skin;

Characterization of oxygenated metabolites of ginsenoside Rg1 in plasma and urine of rat by Jing-Rong Wang; Tian-Tian Tong; Lee-Fong Yau; Cheng-yu Chen; Li-Ping Bai; Jing Ma; Ming Hu; Liang Liu; Zhi-Hong Jiang (75-86).
This study describes the characterization of oxygenated metabolites of ginsenoside Rg1 in rat urine and plasma. These in vivo metabolites were profiled by using UHPLC-QTOF MS-based method. On the basis of high-resolution MS/MS data, and comparison with chemically synthesized authentic compounds, nine oxygenated metabolites of Rg1 were characterized as vinaginsenosides 21 and 22 (M1 and M2), vinaginsenoside R15 (M3), 6-O-(β-d-glucopyranosyl)-20-O-(β-d-glucopyranosyl) 3β, 6α, 12β, 20(S)-tetrahydroxy-24ξ-hydroxydammar-25-ene (M4 and M5), floralginsenoside A (M7 and M8), floralginsenoside B (M9) and epoxyginsenoside Rg1 (M13), respectively. Among these metabolites, M4, M5 and M13 are new ginsenosides and others were detected as in vivo metabolites of Rg1 for the first time. In addition, a series of oxygenated metabolites of Rh1 and deglycosylated metabolite of Rg1, were observed and characterized by comparing with compounds synthesized by us, which revealed an association between C-20 configuration and the extent of oxidation metabolism. Appearance of all these metabolites in blood stream and urine after i.v. dosing and oral administration of Rg1 was further examined, which clearly showed that mono-oxygenated metabolites of Rg1 were major circulating metabolites at the early stage after dosing. Characterization of exact chemical structures of these circulating metabolites contribute greatly to our understanding of chemical exposure after consumption of ginseng products, and provide valuable information for explaining multiple bioactivities of ginseng products.
Keywords: Ginseng; Ginsenoside Rg1; Oxygenation; Metabolites;

A strategy of integrating candidate metabolites with crucial biochemical factors was proposed in this study to discover relevant biological functions for interpreting the action mechanism of Traditional Chinese Medicines (TCM). This approach was applied to Xue-Sai-Tong injection (XST) to reveal the action mechanism based on the metabolic response in an ischemia/reperfusion (I/R) rat model by analyzing NMR profile. Partial least squares discriminate analysis (PLS-DA) was used to compare metabolic profiles of serum samples and revealed nine metabolites altered by I/R injury could be restored to normal status (sham-operated group) under the therapy of XST. The pathway enrichment analysis suggested the metabolic changes were mainly involved in pyruvate metabolism, glycolysis, and citrate cycle. The functional roles of the candidate metabolites were further identified by Pearson correlation analysis with the key biochemical factors in serum. The results indicated pyruvate, succinate, acetate and lysine showed significant associations with the oxidative stress factors. Elevated level of pyruvate was found as an essential metabolic response for the major effect of XST against I/R injury by enhancing glycolysis and overcoming the induced reactive oxygen species (ROS). This metabolomics approach provides a better understanding of the mechanisms of TCM and helps to develop a holistic view of TCM efficacy.
Keywords: Metabolomics; NMR; Non-target; Cardioprotection; Pyruvate;

Determination of pseudoprotodioscin in rat plasma by UPLC–MS/MS: Assay development and application to pharmacokinetic study by Min Liao; Xiao Chen; Jiefeng Chen; Mengping Liu; Junyi Wang; Zuanguang Chen; Zhiyong Xie; Meicun Yao (97-104).
An original and sensitive ultraperformance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method for the determination of pseudoprotodioscin (PPD) in rat plasma was developed and validated. Digitoxin was applied as an internal standard. Plasma samples were processed by acetonitrile-mediated plasma protein precipitation and chromatographed using a step gradient program on a C18 column (2.1 × 50 mm i.d., 1.7 μm). The mobile phase was comprised of acetonitrile and 0.1 mmol L−1 aqueous lithium acetate mixed with 0.03% formic acid at the flow rate of 0.2 mL min−1. Multiple reaction monitoring (MRM) transitions were performed for detection and lithium adduct ions were employed with a significant improvement of the response of the analytes in electrospray positive ionization mode. The concentration range of calibration curve was linear over the range 2–5000 ng mL−1. The intra- and inter-day precisions were all less than 11.5% and accuracies were within the range of 94.1–103.5%, and the analytes exhibited no severe matrix effect. The validated method was successfully applied in the pharmacokinetics of PPD after intragastric (50 mg kg−1) and intravenous (4 mg kg−1) administration in rats. PPD showed rapid excretion and with bioavailability of simply about 5.7% in rats.
Keywords: Pseudoprotodioscin; UPLC–MS/MS; Method validation; Rat plasma; Pharmacokinetics;

Quantitation of eleven active compounds of Aidi injection in rat plasma and its application to comparative pharmacokinetic study by Ran Liu; Ran Ma; Chunyu Yu; Cathy Wenchuan Bi; Yidi Yin; Huarong Xu; Hongwei Shang; Kaishun Bi; Qing Li (105-113).
Aidi injection has been widely used for the treatment of colorectal cancer. The purpose of this study was to develop a sensitive and reliable method for simultaneous quantitation of 11 main active ingredients in Aidi injection and to compare the pharmacokinetics of these ingredients in normal and colorectal model cancer rats after tail vein injection. After being extracted by isopropanol-ethyl acetate (1:1, v/v), the plasma samples were analyzed with domperidone as internal standard. Then the analytes were separated on a Venusil MP C18 column with 0.15% formic acid and methanol. The detection was performed on HPLC–MS/MS system with turbo ion spray source in the positive ion and multiple reaction-monitoring mode. The assay was shown to be linear over the range of 0.004–4.0 μg mL−1 of syringin B, astragaloside II and isofraxidin; 0.01–10.0 μg mL−1 of calycosin-7-O-β-d-glucoside and astragaloside IV; 0.02–20.0 μg mL−1 of ginsenoside Rg1, Rb1, Rc and Rd; 0.04–40.0 μg mL−1 of syringin E; 0.06–60.0 μg mL−1 of ginsenoside Re. And the validated method has been successfully applied to compare pharmacokinetic profiles of the 11 ingredients in plasma. The pharmacokinetic results showed here were significant differences in pharmacokinetic parameters for eight analytes between two groups after injection, while no significant differences for astragaloside II, astragaloside IV and ginsenoside Rc. The present study has the advantages of short analysis time and easy sample preparation, which could more comprehensively reflect the quality of Aidi injection in single run. The method proposed could be of great use for pharmacokinetics, bioavailability or bioequivalence studies of Aidi injection in biological samples.
Keywords: Aidi injection; Colorectal cancer; Pharmacokinetics; Simultaneous determination of multi-components;

A novel chemometrics-assisted high performance liquid chromatography method coupled with diode array detector (HPLC–DAD) was proposed for the simultaneous determination of vincristine (VCR), vinblastine (VLB), vindoline (VDL), catharanthine (CAT) and yohimbine (YHB) in Catharanthus roseus (C. roseus) and human serum samples. With the second-order advantage of the alternating trilinear decomposition (ATLD) method, the resolution and rapid determination of five components of interest in complex matrices were performed, even in the present of heavy overlaps and unknown interferences. Therefore, multi-step purification was omitted and five components could be fast eluted out within 7.5 min under simple isocratic elution condition (acetonitrile/0.2% formic acid water, 37:63, v/v). Statistical parameters, such as the linear correlation coefficient (R 2), root-mean-square error of prediction (RMSEP), limit of detection (LOD) and limit of quantitation (LOQ) had been calculated to investigate the accuracy and reliability of the method. The average recoveries of five vinca alkaloids ranged from 97.1% to 101.9% and 98.8% to 103.0% in C. roseus and human serum samples, respectively. The five vinca alkaloids were adequately determined with limits of detection (LODs) of 29.5–49.3 ng mL−1 in C. roseus and 12.4–27.2 ng mL−1 in human serum samples, respectively. The obtained results demonstrated that the analytical strategy provided a feasible alternative for synchronously monitoring the quality of raw herb and the concentration of blood drugs.
Keywords: Catharanthus roseus herb; Vinca alkaloids; HPLC-DAD; Alternating trilinear decomposition; Second-order calibration;

Scutellariae Radix (SR) has been extensively prescribed in folk medicines due to its notable beneficial activities. The flavonoid glucuronides baicalin (BG), wogonoside (WG), oroxylin A 7-O-β-d-glucuronide (OG) and their aglycones baicalein, wogonin and oroxylin A, are the main components of the herb. So far, majority of previous studies failed to report the pharmacokinetics and none offered an explanation for the systemic exposures of these six flavonoids when the herbal extract was orally administered. In this study, when a SR extract was orally dosed to rats (800 mg/kg, equivalent to BG 324.80, WG 124.00, OG 43.04, baicalein 25.36, wogonin 24.40, and oroxylin A 5.79 mg/kg), all six flavonoids were detectable throughout the experimental period (48 h) using an LC–MS/MS method with the Cmax and AUC0–48 h of the glucuronides 10–130 times that of respective aglycones. As the lowest among the three glucuronides in the herb, OG was the most abundant in vivo, while the systemic exposure of wogonin was the highest amongst the three aglycones. The dose-normalized AUC0–48 h descended in orders of OG/oroxylin A, WG/wogonin and BG/baicalein. Two di-conjugates of baicalein (BG glucuronide and BG glucoside), two BG isomers (minor BM1 and major BM2), and one WG isomer (wogonin 5-O-glucuronide) were detected in rat plasma. Semi-quantitation of the isomers with peak area data revealed that the AUPs (area under peak area ratio-time curves) of BG isomers were ∼3 times that of BG, yet the AUP of wogonin 5-O-glucuronide was only one seventh of WG. BM2, tentatively assigned as baicalein 6-O-glucuronide, was formed from both microbial isomerization of BG and hepatic glucuronidation of baicalein. Wogonin 5-O-glucuronide was only formed in hepatic glucuronidation of wogonin. Demethylated wogonin was observed in gut bacteria, offering an optional origin of BM1 apart from baicalein glucuronidation. Microbial isomerization of BG and extensive hepatic glucuronidation of baicalein to form BM2 as well as a poorer intestinal permeability of baicalein (P app  × 10−6  cm/s) should account for the lower systemic exposures of BG and baicalein. Faster microbial hydrolysis of WG, high intestinal permeability (P app  × 10−5  cm/s) and less hepatic glucuronidation of wogonin explain the relatively high systemic exposure of wogonin. Sole microbial deglycosylation of OG, high intestinal permeability (P app  × 10−5  cm/s) and extensive hepatic glucuronidation of oroxylin A supported the highest systemic exposure of OG. Taken together, the oral kinetics of six flavonoid glucuronides and aglycones in the SR extract were simultaneously obtained. Microbial conversion, intestinal epithelial permeability and hepatic glucuronidation are determinant factors for their systemic exposures.
Keywords: Scutellaria Radix; Systemic exposure; Microbial metabolism; Hepatic glucuronidation; Isomerization; Intestinal permeability;

Metabolism and pharmacokinetics of major polyphenol components in rat plasma after oral administration of total flavonoid tablet from Anemarrhenae Rhizoma by Yuan-yuan Xie; Xiu-ming Wang; Si-huan Wang; Yi-ming Wang; Hui-fang Tian; Yong-sheng Yuan; Hong-yan Li; Qiong-lin Liang; Guo-an Luo (134-144).
Total flavonoid tablet from Anemarrhenae Rhizoma (Zhimu tablet), which was made of total polyphenol components extracted from the dried rhizome of Anemarrhena asphodeloides Bge. (Zhimu in Chinese), is a novel traditional Chinese medicine prescribed for the treatment of diabetes. Mangiferin (MF) and neomangiferin (NMF) are the two main components detected and determined in Zhimu tablet, accounting for 8.9% of the total weight of each tablet. In the present study, high performance liquid chromatography (HPLC) coupled with time-of-flight (TOF) tandem mass spectrometry (MS) was applied to characterize the metabolites of MF and NMF in rat plasmas collected at different time points after oral administration of Zhimu tablet at a dose of 3.63 g/kg (corresponding to 270 mg/kg MF). Accurate mass measurement was used to determine the elemental composition of metabolites and thus to confirm the proposed structures of identified metabolites. Time points of appearance of some metabolites, such as isomers, were also taken into account during the structure confirmation. A total of 21 potential metabolites were found in rat plasma at different time points, and the metabolic pathways in vivo were involved in hydrolysis, methylation, glucuronide conjugation, glycoside conjugation, sulphation, dehydration and isomerisation. Furthermore, a selective and accurate LC–MS assay method was developed and validated for the quantification of MF in plasma. Semi-quantification of main conjugated metabolites was also performed in order to describe the dynamic metabolism profiles of polyphenol components in Zhimu tablet. MF concentration in plasma reached 1.36 ± 0.47 μg mL−1 about 5.0 h after oral administration of Zhimu tablet, which showed a 3.24- and 4.91-fold increase in plasma maximum concentration and area under the concentration-time curve (AUC) from 0 to 24 h of MF compared with those for rats administered with free MF, respectively. The results indicated that the pharmacokinetic processes and bioavailability of MF in rats would be affected by other components in Zhimu tablet.
Keywords: Anemarrhenae rhizoma; Xanthones C-glycosides; Mangiferin; Neomangiferin; Plasma pharmacokinetics; Metabolites; Rat; LC–MS;

Identification of compounds in an anti-fibrosis Chinese medicine (Fufang Biejia Ruangan Pill) and its absorbed components in rat biofluids and liver by UPLC-MS by Qin Dong; Ling-Ling Qiu; Cong-En Zhang; Long-Hu Chen; Wu-Wen Feng; Li-Na Ma; Dan Yan; Ming Niu; Jia-bo Wang; Xiao-he Xiao (145-151).
Liver fibrosis represents a major public health problem worldwide. To date, antifibrotic treatment of fibrosis still remains an unconquered area for western medicine. Fufang Biejia Ruangan Pill (FFBJ) is the first anti-fibrosis drug approved by the China State Food and Drug Administration, and has been demonstrated to have a good antifibrotic efficacy in China. However, the chemical constituents of FFBJ and the absorption and distribution of it in vivo remain unclear, which restricts its research on bioactive components identification and mechanisms of action. In this study, ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) combined with ultra-performance liquid chromatography/triple quadrupole mass spectrometry (UPLC/QqQ-MS) was applied to identify compounds in FFBJ and its absorbed components in rat serum, liver and urine samples after intragastric administration of FFBJ. As a result, a total of 32 Chinese material medica components including organic acids, terpenoids, flavonoids, phenylpropanoids and alkaloids, were identified or tentatively characterized, while the distribution of 10 prototype compounds in rat serum, liver and urine were discovered. The identified constituents in FFBJ and the distribution of prototype compounds in rat serum, liver and urine are help for understanding the material bases of its therapeutic effects.
Keywords: Fufang Biejia Ruangan Pill; UPLC/Q-TOF-MS; UPLC/QqQ-MS; Absorption; Distribution;

The herbal preparation Ma-Xing-Gan-Shi-Tang (MXGST) is a popular traditional Chinese formulation that has been used for the treatment of coughs and fevers. The potential active components of MXGST are ephedrine, amygdalin, and glycyrrhizic acid. The aim of this study was to develop a validated analytical method to measure these analytes in the herbal preparation MXGST using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS). Multiple reaction monitoring (MRM) was used to monitor m/z 166.1 → 148.1 for ephedrine ([M+H]+), 475.2 → 163.0 for amygdalin ([M + NH4]+), and 840.6 → 453.3 ([M+NH4]+) for glycyrrhizic acid. The analytes were separated by a reverse phase C18 column (100 × 2.1 mm, 2.6 μm). The mobile phase consisted of 5 mM ammonium acetate (0.1% formic acid) and 100% methanol (0.1% formic acid) with a linear gradient elution. Five brands of commercial pharmaceutical herbal products and a laboratory extract of MXGST were analyzed. Moreover, the modified UHPLC–MS/MS method was applied to the comparative pharmacokinetics of ephedrine in rats from the following three sources: (1) pure ephedrine, (2) an herbal extract of Ephedra, and (3) an herbal preparation of MXGST. Plasma samples from rats were prepared by protein precipitation, evaporation and reconstitution. The pharmacokinetic data showed that pure ephedrine was absorbed significantly faster than ephedrine of the Ephedra extract or the MXGST herbal preparation. However, the elimination half-life of ephedrine administered as the pure compound was 93.9 ± 8.07 min, but for ephedrine from the Ephedra extract and the MXGST, the half-lives were 133 ± 17 and 247 ± 57.6 min, respectively. The area under the concentration curves (AUC) did not show significant differences among the three groups. These data suggest that the rest of the herbal ingredients in the Ephedra extract and the MXGST may provide a compensation effect that reduces the peak concentration of ephedrine and prolongs the elimination half-life.
Keywords: Traditional Chinese medicine; Ephedra extract; Ephedrine; Amygdalin; Glycyrrhizic acid; Pharmacokinetics;

The pharmacokinetic characters of simvastatin after co-administration with Shexiang Baoxin Pill in healthy volunteers’ plasma by Jianfei Tao; Peng Jiang; Chengcheng Peng; Min Li; Runhui Liu; Weidong Zhang (162-167).
To investigate the effect of Shexiang Baoxin Pill (SBP), a tranditional Chinese medicine, on the pharmacokinetic (PK) parameters of simvastatin in healthy volunteers’ plasma, a quantitative method was developed using an Agilent G6410A rapid performance liquid chromatography (RPLC) coupled with triple quadrupole mass spectrometry system. The established method was rapid with high extraction recovery and successfully applied for the determination of simvastatin in plasma of 16 healthy volunteers. The results demonstrated that the MRT(0–∞), T 1/2 and T max value of simvastatin were significantly decreased, while the AUC(0–t) and C max values of smivastatin were increased by SBP. The pharmacokinetic study demonstrated that the metabolism parameters of simvastatin could be affected by SBP and the potential drug–drug interaction should be noted in the future clinical practice.
Keywords: Simvastatin; Shexiang Baoxin Pill; Pharmacokinetics; Healthy volunteers;

A metabolomics strategy to explore urinary biomarkers and metabolic pathways for assessment of interaction between Danhong injection and low-dose aspirin during their synergistic treatment by Jianping Li; Jianming Guo; Erxin Shang; Zhenhua Zhu; Kevin Yue Zhu; Shujiao Li; Buchang Zhao; Lifu Jia; Jing Zhao; Zhishu Tang; Jinao Duan (168-175).
The drug combination of Danhong injection (DHI) and low-dose aspirin (ASA) was frequently applied for the treatment of cardiovascular and cerebrovascular diseases. Due to the drug interactions, a lot of potential benefits and risks might exist side by side in the course of combination therapy. However, there had been no studies of interaction between DHI and ASA. Metabolomics was a powerful tool to explore endogenous biomarkers and metabolic pathways. In present study, metabolic profiling with ultra-high-performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UHPLC-QTOF/MS) coupled with multivariate statistical analysis was performed to provide insight into understanding the interaction between DHI and low-dose ASA. Eleven potential biomarkers of three types were identified and seven metabolic pathways were constructed. The results showed that the interaction between DHI and low-dose ASA during synergistic treatment indeed affected some key endogenous biomarkers and metabolic pathways, which could not happen when DHI or low-dose ASA was used alone. The quality and quantity of endogenous metabolite were both influenced by interaction between DHI and low-dose ASA. In details, the amount of flavin mononucleotide, L-2, 4-diaminobutyric acid (DABA) and 4-aminohippuric acid were significantly increased. On the contrary, the amount of 3-methyluridine, 4, 6-dihydroxyquinoline, cortolone-3-glucuronide, and serotonin were significantly decreased. Furthermore, O-phosphotyrosine, 3-methyl-2-butenal, indoxyl sulfate and dolichyl diphosphate were disappeared in urine. As to metabolic pathways, riboflavin metabolism, pentose and glucuronate interconversions, and tryptophan metabolism were all significantly influenced. The emerging alterations of biomarkers and metabolic pathways were associated with a lot of drugs and diseases based on literature researches, which might influence the co-administration of other drugs or the treatments of relevant diseases. Our paper presented some hints to uncover the mechanism of interaction between DHI and low-dose ASA, which would provide some references for application of DHI and low-dose ASA combination.
Keywords: Interaction; Metabolomics; Drug combination; Danhong injection; Aspirin;

Metabolic changes at the early stage of sepsis induced by cecal ligation and puncture in rats and the interventional effects of Huang-Lian-Jie-Du-Tang by Yufen Wei; Na Gao; Zhongxiao Zhang; Xianpeng Zu; Zhenlin Hu; Weidong Zhang; Jun Yin; Xinru Liu (176-182).
Sepsis is a disease with high mortality that requires rapid diagnosis and treatment. This study used a metabolomic approach to profile the metabolic changes at the early stage of sepsis induced by cecal ligation and puncture (CLP) in rats and investigated the interventional effects of Huang-Lian-Jie-Du-Tang (HLJDT). Male SD rats were intragastrically administered 270 mg/kg HLJDT 2 h prior to CLP, serum extracts were profiled by liquid chromatography/quadrupole time-of-flight mass spectrometer (LC–Q-TOF-MS) and multivariate analytical (MVA) methods were employed to evaluate the metabolic changes of extracts. A Partial Least-Squares Discriminant Analysis (PLS-DA) score plot indicated that septic rats undergo significant metabolic changes 2 h after CLP, and HLJDT administration could reverse the metabolic changes induced by CLP. Sixteen biomarkers involved in amino acid metabolism, unsaturated fatty acid metabolism, purine metabolism, and lipid metabolism were identified after Orthogonal Partial Least-Squares Analysis (OPLS). Among the 16 metabolites, 10 were regulated by HLJDT. This study established the foundation for further research of the early diagnosis biomarkers and therapeutic evaluation biomarkers discovery of sepsis.
Keywords: Metabolic profiling; Sepsis; Cecal ligation and puncture; Huang-Lian-Jie-Du-Tang;

This research was designed to study metabonomic characteristics of the toxicity induced by yuanhuapine, a major bioactive diterpenoid in a well-known traditional Chinese medicine-Genkwa Flos. General observation, blood biochemistry and histopathological examination were used to reflect yuanhuapine-induced toxicity. Urine samples from rats in control and yuanhuapine treated rats were analyzed by ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC–Q-TOF/MS). Pattern recognition methods including principal components analysis (PCA), partial least-squared discriminant analysis (PLS-DA), orthogonal partial least-squared discriminant analysis (OPLS-DA) and computational system analysis were integrated to obtain comprehensive metabonomic profiling and pathways of the biological data sets. The results suggested that yuanhuapine could induce intestinal and liver damage. And 14 endogenous metabolites as biomarkers related to the amino acids metabolism, lipids metabolism, carbohydrate metabolism and gut microflora were significantly changed in the urine of yuanhuapine treated rats, which were firstly constructed the metabolomic feature profiling and metabolite interaction network of yuanhuapine-induced injury using pattern recognition methods and Ingenuity Pathway Analysis (IPA) approach. The present study showed that yuanhuapine-induced intestinal and hepatic toxicity were correlated with disturbance of amino acids metabolism, lipids metabolism, carbohydrate metabolism and gut microflora.
Keywords: Yuanhuapine; Genkwa Flos; Metabonomic profile; Toxicity;

Colon-derived uremic biomarkers induced by the acute toxicity of Kansui radix: A metabolomics study of rat plasma and intestinal contents by UPLC-QTOF-MSE by Zhou Yang; Jin-Jun Hou; Peng Qi; Min Yang; Bing-Peng Yan; Qi-Rui Bi; Rui-Hong Feng; Wen-Zhi Yang; Wan-Ying Wu; De-An Guo (193-203).
Kansui radix (KR) is a poisonous Chinese herbal medicine recorded in the Chinese Pharmacopoeia, and the acute toxicity obstructs its clinical applications. To explore its acute toxicity mechanism to enhance clinical safety, a metabolomics study based on UPLC-ESI-QTOF-MSE was performed. Wistar rats were exposed for 4 h to the aqueous and ethyl acetate extracts prepared from KR at a high dose (25 g/kg). The contents of six different sections of rat intestine, including the duodenum, jejunum, ileum, cecum, colon, and rectum were collected as samples for the first time, as well as the rat plasma. The interesting results showed that only those rats exposed to the ethyl acetate extract showed a watery diarrhea, similar to the observed acute human toxicity. The identified biomarkers found in the plasma, such as phenol sulfate, indoxyl sulfate, and p-cresol sulfate were significantly perturbed in the rats. These biomarkers are known as colon-derived uremic compounds, which were first reported with respect to KR. The three essential amino acids which produced these biomarkers were only found in the contents of colon and rectum. A hypothesis was proposed that only the colon-derived uremic compounds induced by KR might be responsible for the acute toxicity. Three traditional process methods to reduce the toxicity of KR were compared based on these biomarkers, and different levels of toxicity modulation were observed. These results may be helpful to further understand the mechanism of acute toxicity, and the relevance of the traditional process methods to ameliorate the adverse effects of KR.
Keywords: Kansui radix; Metabolomics; Toxicity; Intestinal contents; Colon-derived uremic biomarkers;

Metabolomics and its application to the evaluation of the efficacy and toxicity of traditional Chinese herb medicines by Jian Shi; Bei Cao; Xin-Wen Wang; Ji-Ye Aa; Jin-Ao Duan; Xuan-Xuan Zhu; Guang-Ji Wang; Chang-Xiao Liu (204-216).
Traditional Chinese herb medicines (TCHMs) have been used in the treatment of a variety of diseases for thousands of years in Asian countries. The active components of TCHMs usually exert combined synergistic therapeutic effects on multiple targets, but with less potential therapeutic effect based on routine indices than Western drugs. These complex effects make the assessment of the efficacy of TCHMs and the clarification of their underlying mechanisms very challenging, and therefore hinder their wider application and acceptance. Metabolomics is a crucial part of systems biology. It allows the quantitative measurement of large numbers of the low-molecular endogenous metabolites involved in metabolic pathways, and thus reflects the fundamental metabolism status of the body. Recently, dozens of metabolomic studies have been devoted to prove the efficacy/safety, explore the underlying mechanisms, and identify the potential biomarkers to access the action targets of TCHMs, with fruitful results. This article presents an overview of these studies, focusing on the progress made in exploring the pharmacology and toxicology of various herbal medicines.
Keywords: Metabolomics; Drug efficacy; Toxicity; Traditional Chinese herb medicines;

Serum metabolomics strategy for understanding pharmacological effects of ShenQi pill acting on kidney yang deficiency syndrome by Yang Nan; Xiaohang Zhou; Qi Liu; Aihua Zhang; Yu Guan; Shanhua Lin; Ling Kong; Ying Han; Xijun Wang (217-226).
Kidney yang deficiency syndrome, a diagnostic pattern in Chinese medicine, is similar with clinical features of the glucocorticoid withdrawal syndrome. The aim of this present study was to explore low molecular mass differentiating metabolites between control group and model group of kidney yang deficiency rats induced with corticosterone as well as the therapeutic effect of Shen Qi Pill, a classic traditional Chinese medicine formula for treating Kidney yang deficiency syndrome in China. This study utilized ultra-performance liquid chromatography coupled with electrospray ionization synapt quadrupole time-of-flight high definition mass spectrometry (UPLC/ESI–SYNAPT–QTOF–HDMS) to identify the underlying biomarkers for clarifying mechanism of Shen Qi Pill in treating Kidney yang deficiency syndrome based on metabolite profiling of the serum samples and in conjunction with multivariate and pathway analysis. Meanwhile, blood biochemistry assay and histopathology were examined to identify specific changes in the model group rats. Distinct changes in the pattern of metabolites were observed by UPLC–HDMS. The changes in metabolic profiling were restored to their baseline values after treatment with Shen Qi Pill according to the combined with a principal component analysis (PCA) score plots. Altogether, the current metabolomics approach based on UPLC–HDMS and orthogonal projection to latent structures discriminate analysis (OPLS–DA) demonstrated 27 ions (18 in the negative mode, 9 in the positive mode, 17 ions restored by Shen Qi Pill). These results indicated that effectiveness of Shen Qi Pill in Kidney yang deficiency syndrome rats induced a substantial change in the metabolic profiles by regulating the biomarkers and adjusting the metabolic disorder. It suggested that the metabolomics approach was a powerful approach for elucidation of pathologic changes of Chinese medicine syndrome and action mechanisms of traditional Chinese medicine.
Keywords: Metabolomics; Kidney yang deficiency syndrome; Metabolites; Biomarkers; Shen Qi pill; Pattern recognition approaches; UPLC–ESI–Q–TOF–HDMS;

A GC–MS urinary quantitative metabolomics analysis in depressed patients treated with TCM formula of Xiaoyaosan by Jun-Sheng Tian; Guo-Jiang Peng; Yan-Fei Wu; Jian-Jun Zhou; Huan Xiang; Xiao-Xia Gao; Yu-Zhi Zhou; Xue-Mei Qin; Guan-Hua Du (227-235).
Xiaoyaosan, one of the best-known traditional Chinese medicine prescriptions, has been widely used in China for the treatment of mental disorders such as depression. Although both clinical application and animal experiments indicate that Xiaoyaosan has an obvious antidepressant effect, the mechanism still remains unclarified, and there are few studies quantitatively measured the biomarkers of Xiaoyaosan treatment by metabolomics to determination. In this study, 25 depressed patients and 33 healthy volunteers were recruited. A GC–MS based metabolomics approach and the multivariate statistical methods were used for analyzing the urine metabolites of depressed patients before and after treatment compared with healthy controls. Then the biomakers through metabolomics determination were carried out the quantitative analysis. In total, 5 metabolites were identified as the potential diseased and therapeutic biomarkers of depression and Xiaoyaosan. Alanine, citrate and hippurate levels were significantly increased in the urine samples from depressed patients compared with healthy controls, while phenylalanie and tyrosine levels were significantly decreased. However, after Xiaoyaosan treatment for 6 weeks, phenylalanie and tyrosine levels were significantly increased (p  < 0.05) and alanine, citrate and hippurate levels significantly decreased (p  < 0.05). Xiaoyaosan has a good priority on the treatment of depression and the ability to adjust the neurotransmitters to obtain the best treated response and also could regulate the metabolism of amino acids and promote to produce energy meet the needs of the body.
Keywords: Xiaoyaosan; Gas chromatography–mass spectrometry (GC–MS); Depression; Metabolomics; Quantitative analysis;

The increasing use of Chinese herbal medicines (CHMs) as complementary therapy and dietary supplement has been greatly raising the concerns about potential herb–drug interactions (HDIs). HDIs may cause the augmented or antagonized effects of prescription drugs, resulting in unexpected clinical outcomes. Therefore, it is of significance to identify or predict potential HDIs, and to delineate the underlying mechanisms. Drug transporters play key roles in transmembrane passage of a large number of drugs, affecting their absorption, distribution and elimination. Modulation of drug transporters has been recognized as one of the main causes of HDIs. In the last decade, a growing number of Chinese medicinal herbs and their derived phytochemicals have been identified to have modulatory effect toward transporter proteins, leading to pharmacokinetic HDIs when concomitantly used with conventional drugs. Some of these transporter-mediated interactions have already shown clinical significance. This review article focuses on two major transporter superfamilies, the solute carrier (SLC) and the ATP-binding cassette (ABC) transporters, to provide the recent advanced knowledge on CHMs and their inherent phytochemicals that interact with these transporters, and their induced pharmacokinetic HDIs from both preclinical and clinical aspects. In addition, the challenges and strategy for studying HDIs are also discussed.
Keywords: Herb–drug interaction; Solute carrier transporter; ATP-binding cassette transporter; Chinese herbal medicines; Natural products;

Chemical and biological assessment of Jujube (Ziziphus jujuba)-containing herbal decoctions: Induction of erythropoietin expression in cultures by Candy T.W. Lam; Pui H. Chan; Pinky S.C. Lee; Kei M. Lau; Ava Y.Y. Kong; Amy G.W. Gong; Miranda L. Xu; Kelly Y.C. Lam; Tina T.X. Dong; Huangquan Lin; Karl W.K. Tsim (254-262).
Jujubae Fructus, known as jujube or Chinese date, is the fruit of Ziziphus jujuba (Mill.), which not only serves as daily food, but acts as tonic medicine and health supplement for blood nourishment and sedation. According to Chinese medicine, jujube is commonly included in herbal mixtures, as to prolong, enhance and harmonize the efficiency of herbal decoction, as well as to minimize the toxicity. Here, we aim to compare the chemical and pharmacological properties of three commonly used jujube-containing decoctions, including Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and Zao Tang (ZOT). These decoctions share common herbs, i.e. Glycyrrhizae Radix et Rhizoma Praeparata cum Melle, Zingiberis Rhizoma Recens and Jujube, and they have the same proposed hematopoietic functions. The amount of twelve marker biomolecules deriving from different herbs in the decoctions were determined by LC–MS, and which served as parameters for chemical standardization. In general, three decoctions showed common chemical profiles but with variations in solubilities of known active ingredients. The chemical standardized decoctions were tested in cultured Hep3B cells. The herbal treatment stimulated the amount of mRNA and protein expressions of erythropoietin (EPO) via the activation of hypoxia response elements: the three herbal decoctions showed different activation. The results therefore demonstrated the hematopoietic function of decoctions and explained the enhancement of jujube function within a herbal mixture.
Keywords: TCM; Decoctions; Erythropoietin; Jujube; LC–DAD–MS/MS;

Localization of ginsenosides in Panax ginseng with different age by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry imaging by Hangrui Bai; Shujuan Wang; Jianjun Liu; Dan Gao; Yuyang Jiang; Hongxia Liu; Zongwei Cai (263-271).
The root of Panax ginseng C.A. Mey. (P. ginseng) is one of the most popular traditional Chinese medicines, with ginsenosides as its main bioactive components. Because different ginsenosides have varied pharmacological effects, extraction and separation of ginsenosides are usually required for the investigation of pharmacological effects of different ginsenosides. However, the contents of ginsenosides vary with the ages and tissues of P. ginseng root. In this research, an efficient method to explore the distribution of ginsenosides and differentiate P. ginseng roots with different ages was developed based on matrix assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF-MSI). After a simple sample preparation, there were 18 peaks corresponding to 31 ginsenosides with distinct localization in the mass range of m/z 700–1400 identified by MALDI-TOF-MSI and MALDI-TOF-MS/MS. All the three types of ginsenosides were successfully detected and visualized in images, which could be correlated with anatomical features. The P. ginseng at the ages of 2, 4 and 6 could be differentiated finely through the principal component analysis of data collected from the cork based on the ion images but not data from the whole tissue. The experimental result implies that the established method for the direct analysis of metabolites in plant tissues has high potential for the rapid identification of metabolites and analysis of their localizations in medicinal herbs. Furthermore, this technique also provides valuable information for the component-specific extraction and pharmacological research of herbs.
Keywords: Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry imaging; Panax ginseng; Ginsenosides; Age; Differentiation;

Morbidity of hyperuricemia has constantly increased in population in decades, and hyperuricemia has proved to be an important risk factor for gout, cardiovascular diseases and others. Many urate-lowering drugs have unfavorable side effects and drug interactions. Quzhuotongbi decoction (QZTBD) is an empirical traditional Chinese medicine prescription for clinical therapy of hyperuricemia without serious adverse effects. In the study, we investigated the effects of QZTBD on urate and other metabolites in the sera of hyperuricemia model rats. Hyperuricemia model was established by orally administering yeast extract paste, and allopurinol served as a positive control drug. Serum metabolomics was performed by using a gas chromatography–mass spectrometry (GC–MS) method. Student’s t-test and the principal component analysis (PCA) were employed to find the metabolic perturbations in hyperuricemia model rats. The levels of urate, lactate, pyruvate and ornithine were significantly increased, and xanthine, glyconic acids (ribonate, galactonate), amino acids (aspartate, proline, glutamine, serine, pyroglutamate, glutamate) and glucose were down-regulated greatly in the model rats. It demonstrated that nucleotide metabolism, amino acid metabolism and glycolytic pathway were disturbed by yeast administration. An orthogonal signal correction-partial least-squares discriminant analysis (OSC-PLS DA) was performed to assess the effects of yeast administering and drug treatment. 11 significantly distinctive metabolites among four groups were defined according to the variable importance for project values (VIP > 1) and univariate ANOVA (p value < 0.05). As compared to the model rats, the serum uric acid levels were lowered markedly under the treatment of allopurinol or QZTBD. Aspartate and glutamine involved in purine metabolism, were raised to normal level as well. The different influences on xanthine, glutamate pyroglutamate and galactonate suggested there were different mechanisms of two drugs in urate-lowering therapy. Our finding proved that QZTBD can efficiently lower the level of serum uric acid in a different way from allopurinol, which suggested that QZTBD based on the theory of TCM could be an effective therapeutic option for hyperuricemia.
Keywords: Hyperuricemia; TCM; Metabolomics; Serum; GC–MS; Uric acid;

Current approaches and challenges for chemical characterization of inhibitory effect against cancer cell line isolated from Gokshur extract by Salwa Bouabdallah; Rabiaa-M. Sghaier; Sawssen Selmi; Daycem Khlifi; Dhafer Laouini; Mossadok Ben-Attia (279-285).
In the present study, the potential effect anti tumor and the chemical composition of different fractions of Gokshur was evaluated. Commonly known as puncture vine, it has been used for a long time in both the Indian and traditional Chinese medicine. It is popularly used as a remedy for fertility disorder in Ayurveda. Samples were collected during June–September 2014 in the Cap Bon (north east of the northern Tunisia). Different organs were separated and extracted by sequential process to compare and investigate their potential anti-tumor effect. For the first time, we report the antiproliferatif effect of leaves n-butannolic fraction against cancer cell lines. The better anti-tumor fraction (94.76 ± 1.52%) has been detected and performed by RP-HPLC has shown a great peak area (5578.21 Mau). Novel designed natural derivatives from Gokshur, a cyclotrisiloxane, major compound identified by GC–MS.
Keywords: Anti-cancer; ATCM; GC–MS; Cyclotrisiloxane; Gokshur; MTTassay; RP-HPLC;