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A novel "Prediction and Confirmation" (PC) strategy was proposed for characterizing phosphodiesterase-5 inhibitor (PDE-5) derivatives in botanical dietary supplements (BDSs) for on-site detection. Discovery Studio (DS) and density functional theory (DFT) calculations were used for the "Prediction" step in order to estimate PDE-5 derivative structures and theoretical Raman shifts without synthesizing the derivatives. After 11 potentially bioactive sildenafil derivatives were acquired through DS, 32 common calculated Raman shifts were obtained through DFT. The mean absolute wavenumber deviation (δ, peak range) of the major bands and the minimum number (τ) of Raman spectral peaks matching the calculated common shifts were optimized, so that a positive result of an unknown sample could be reasonably produced. In this study, δ was set at ±10 cm -1 and the corresponding τ was set at 4-5 after optimization. Surface plasmon resonance (SPR) biosensor and surface-enhanced Raman scattering (SERS) detection were the "Confirmation" step to validate the reliability and accuracy of DS and DFT in the "Prediction" step, respectively. The optimized δ and τ criteria were used as indexes for on-site SERS detection after thin-layer chromatographic (TLC) separation of six real-world samples, one of which was preliminarily identified as "suspected positive samples." This strategy allows for a quick determination of the BDSs adulterated with sildenafil or its derivatives, independent of any standard materials.

A surface plasmon resonance (SPR) biosensor-based active ingredients recognition system (SPR-AIRS) was developed, validated, and applied to screen signal transducer and activator of transcription 3 (STAT3) ligands. First, features of the screening system were investigated in four aspects: (1) specificity of the STAT3-immobilized chip, it shows that the chip could be applied to screen STAT3 ligands from complex mixture; (2) linearity and limit of detection (LOD) of the system, the minimum recovery cycle number was determined as 5 cycles; (3) saturability of the chip, the results indicate that it is necessary to select a proper concentration based on the compound's K d value; (4) robustness of the system, it indicates that inactive compounds in the matrix could not interfere with active compounds in the process of screening. Next, SPR-AIRS was applied to screen STAT3 ligands from medicinal herbs. Nine candidate compounds were fished out. Then SPR assay and molecular docking were performed to verify the interplay between STAT3 and candidate compounds. Apoptosis assay and luciferase report assay were performed to investigate the drug effect of candidate compounds on STAT3 activity. Western blot results indicated that neobaicalein and polydatin could inhibit the phosphorylation of STAT3. As far as we know, this is the first time that neobaicalein and polydatin are reported as effective STAT3 ligands. In a conclusion, we have systemically demonstrated the feasibility of SPR biosensor-based screening method applying to complex drug systems, and our findings suggest that SPR-AIRS could be a sensitive and effective solution for the discovery of active compounds from a complex matrix.

Cell membrane chromatography (CMC) is an ideal method for screening potential active components acting on target cell membranes from a complex system, such as herbal medicines. But due to the decay and falling-off of membranes, the CMC column suffers from short life span and low reproducibility. This has greatly limited the application of this model, especially when the cell materials are hard to obtain. To solve this problem, a novel type of (3-aminopropyl)triethoxysilane (APTES)-decorated silica gel was employed. The silica gel was decorated with aldehydes with the help of APTES, which react with the amino groups on cell membranes to form a covalent bond. In this way, cell membranes were immobilized on the surface of silica gel, so it is not easy for membranes to fall off. According to our investigation, the column life of the APTES-decorated group was prolonged to more than 12 days, while the control group showed a sharp decline in column efficiency in the first 3 days. To verify this model, a novel APTES-decorated HepG2 cancer stem cell membrane chromatography (CSCMC) was established and applied in a comprehensive two-dimensional chromatographic system to screen potential active components in Salvia miltiorrhiza. As a result, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I were retained on this model and proved to be effective on HepG2 cancer stem cells by the following cell proliferation and apoptosis assay, with IC 50 of 10.30 μM, 17.85 μM, and 2.53 μM, respectively. This improvement of CMC can significantly prolong its column life span and broaden the range of its application, which is very suitable for making invaluable or hard-to-obtain cell materials, such as stem cells, for specific drug screening.

Sini decoction (SND) is an important traditional Chinese multiherbal formula, which is widely used to treat cardiovascular disease. Acontium carmichaeli (AC) is a leading herb in SND, whose main components are monoester-diterpenoid alkaloids (MDAs). The aim of this study is to compare the pharmacokinetics of three MDAs in rat plasma after oral administration of AC extract and its compatibility with other herbal medicines in SND. A sensitive, accurate and specific LC-MS/MS method was developed to determine the contents of three MDAs in rat plasma. Male Sprague-Dawley rats were randomly assigned to four groups: AC, AC + ZO, AC + GU and SND groups. There were significant differences in the pharmacokinetic parameters (Cmax, Tmax, t1/2, AUC(0-24), MRT and CL). Compared with the AC group, Cmax, AUC(0-24) and CL of three MDAs increased and t1/2 decreased in AC + ZO, AC + GU and SND groups. Little changed in the AC + GU group in comparison with AC + ZO group, which indicated that other ingredients in ZO may promote the absorption rate and accelerate excretion rate of MDAs. The results could be helpful for revealing the compatibility mechanism of Chinese multiherbal medicine and providing clinical medication guidance on AC and its compatibility with other herbal medicines in SND., (Copyright © 2014 John Wiley & Sons, Ltd.)

Cell membrane chromatography (CMC) derived from pathological tissues is ideal for screening specific components acting on specific diseases from complex medicines owing to the maximum simulation of in vivo drug-receptor interactions. However, there are no pathological tissue-derived CMC models that have ever been developed, as well as no visualized affinity comparison of potential active components between normal and pathological CMC columns. In this study, a novel comparative normal/failing rat myocardium CMC analysis system based on online column selection and comprehensive two-dimensional (2D) chromatography/monolithic column/time-of-flight mass spectrometry was developed for parallel comparison of the chromatographic behaviors on both normal and pathological CMC columns, as well as rapid screening of the specific therapeutic agents that counteract doxorubicin (DOX)-induced heart failure from Acontium carmichaeli (Fuzi). In total, 16 potential active alkaloid components with similar structures in Fuzi were retained on both normal and failing myocardium CMC models. Most of them had obvious decreases of affinities on failing myocardium CMC compared with normal CMC model except for four components, talatizamine (TALA), 14-acetyl-TALA, hetisine, and 14-benzoylneoline. One compound TALA with the highest affinity was isolated for further in vitro pharmacodynamic validation and target identification to validate the screen results. Voltage-dependent K(+) channel was confirmed as a binding target of TALA and 14-acetyl-TALA with high affinities. The online high throughput comparative CMC analysis method is suitable for screening specific active components from herbal medicines by increasing the specificity of screened results and can also be applied to other biological chromatography models.

The combined use of the state-of-the-art hybrid mass spectrometer (MS) together with high efficient liquid chromatography may prove to be a useful tool for neutral saccharide analysis. In the present work, we used hydrophilic interaction liquid chromatography (HILIC) to separate selected saccharides (fructose, glucose, galactose, sucrose, melibiose, raffinose, manninotriose, stachyose and verbascose). Influences of the column type, additive type, temperature, pH and other separation factors on analyte retention were evaluated. The new method did not require reduction, derivatization or postcolumn addition of reagents, which are commonly used in conventional saccharide analysis. Our results demonstrate the potential of HILIC-MS for sensitive and robust determination of saccharides in crude and processed Radix Rehmanniae, and may promote new perspectives in the research of other medicinal herbs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the work presented here, a novel approach to comprehensive two-dimensional liquid chromatography is evaluated. Reversed-phase liquid chromatography was employed for the first-dimension separation and polyamine chromatography was chosen for the second-dimension separation mode. The two dimensions are highly orthogonal and the separation efficacy of the developed octadecylsilica × polyamine was tested by separating an extract from Anemarrhena asphodeloides. The steroid glycosides identified by comprehensive two-dimensional liquid chromatography in this experiment were compared to those obtained for monodimensional liquid chromatography. The comprehensive two-dimensional liquid chromatography system, thanks to the complementary separation selectivity and enhanced peak capacity provided by the two columns, allowed to distribute five compounds of low amounts otherwise unachievable by monodimensional liquid chromatography. In addition, four steroid isomers with similar fragmentation characteristics in MS/MS spectra, were newly separated based on their different chemical structures., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This work describes a novel methodology for unequivocal identification of chemical constituents in Zhimu-Baihe herb-pair (ZMBHHP). Compounds were removed from ZMBHHP by ultrasonic extraction with 70% ethanol, and then analyzed by fast high-performance liquid chromatography (HPLC) coupled with time-of-flight mass spectrometry (TOFMS). The accurate-mass capability of the TOF analyzer allowed reliable confirmation of the identity of the detected compounds, normally with mass errors below 3 ppm in routine analysis. This mass accuracy was sufficient to verify the elemental compositions of the chemical constituents in ZMBHHP. With dynamic adjustment of fragmentor voltage in TOFMS, an efficient ion transmission was achieved to obtain the best sensitivity and abundant fragmentation. By accurate mass measurements for each molecular ion and subsequent fragment ions, a reliable identification and differentiation of 24 saponins, 3 xanthones, 1 anthraquinone and 2 alkaloids was described here, including four groups of isomers. It is concluded that this fast and sensitive HPLC/ESI-TOFMS technique is powerful in qualitative analysis of complex herbal medicines in terms of sensitivity, selectivity, resolving power, time savings and lower solvent consumption. Furthermore, the data gathered in this study may be helpful for understanding the synergistic nature of this herb pair in traditional Chinese medicine (TCM) and further pharmacokinetic studies of ZMBHHP., (Copyright © 2010 John Wiley & Sons, Ltd.)

Candida albicans is the most important human fungal pathogen. Amphotericin (AmB) is a gold standard of antifungal treatment for fungi, but the severe side effect of this drug restricts its clinical application. In this study, the interaction of AmB and baicalein (BE) was investigated against thirty clinical isolates of C. albicans. Synergistic activities were determined using the checkerboard microdilution assay based on the fractional inhibitory concentration indices. Combination of BE and AmB accelerated C. albicans apoptosis accompanied with an increase of reactive oxygen species (ROS). Moreover, AmB increased the caspase activity and expression of the corresponding gene CaMCA1 in C. albicans. These effects were enhanced in the presence of BE. Deletion of CaMCA1 clearly attenuated AmB-induced apoptosis, indicating the involvement of CaMCA1-mediated caspase pathway in AmB-induced apoptosis and the synergistic action.

Shuang-Dan (SD) is a traditional Chinese prescription containing Cortex Moutan and Radix Salviae miltiorrhizae and commonly used for treating cardiovascular disease. Paeoniflorin is a main effective ingredient of Cortex Moutan and the pharmacokinetic differences of paeoniflorin following oral administration of pure paeoniflorin, Cortex Moutan extract and SD decoction to rats were studied with approximately the same dose of 30mg/kg paeoniflorin. At different time points (5, 10, 15, 30, 45, 60, 90, 120, 150, 210, 270, 360, 450min), plasma concentration of paeoniflorin was determined using a simple and rapid HPLC-MS method. Unpaired student's t-test was used for the statistical comparison. A bimodal phenomenon was observed in the plasma profile after oral administration of Cortex Moutan extract. Statistically significant increase (P<0.05) in pharmacokinetic parameters of paeoniflorin including AUC(0-t), AUC(0-infinity) and MRT were obtained after oral administration of Cortex Moutan or SD decoction comparing with pure paeoniflorin. The investigation showed that among all calculated parameters, AUC(0-t), AUC(0-infinity), MRT, k(e) and T(1/2), there were no significant differences between the two decoctions. The results indicated that the reason which delay the elimination of paeoniflorin and enhance its bioavailability might be some ingredients in Cortex Moutan extract.

Abstract: The aim of this study was to compare the pharmacokinetics of baicalin and wogonoside in rats following oral administration of Xiaochaihu Tang (Minor Radix Bupleuri Decoction) and Radix scutellariae extract. Thus, a specific LC–MS method was developed and validated for the determination of these flavonoids in the plasma of rats after oral administration Xiaochaihu Tang and Radix scutellariae extract. Chromatographic separation was performed on a Zorbax SB C18 column (150mm×4.6mm, i.d.: 5μm) with 0.1% formic acid in water and acetonitrile by linear gradient elution. Baicalin, wogonoside and carbamazepine (internal standard, I.S.) were detected in select-ion-monitoring (SIM) mode with a positive electrospray ionization (ESI) interface. The following ions: m/z 447 for baicalin, m/z 461 for wogonoside and m/z 237 for the I.S. were used for quantitative determination. The calibration curves were linear over the concentration ranges from 0.1231 to 6.156μgmL−1 for baicalin and 0.08832 to 4.416μgmL−1 for wogonoside. The lower limit of detection (LLOD) based on a signal-to-noise ratio of 2 was 0.06155μgmL−1 for baicalin and 0.04416μgmL−1 for wogonoside. Intra-day and inter-day precisions (RSD%) were within 10% and accuracy (RE%) ranged from −6.4 to 4.4%. The extraction recovery at three QC concentrations ranged from 74.7 to 86.0% for baicalin and from 71.3 to 83.7% for wogonoside. The plasma concentrations of baicalin and wogonoside in rats at designated time periods after oral administration were successfully determined using the validated method, pharmacokinetic parameters were estimated by a non-compartment model. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t 1/2 of baicalin was 3.60±0.90 and 5.64±1.67, the C max1 was 1.64±0.99 and 5.66±2.02, the t max1 was 0.13±0.05 and 0.20±0.07, the C max2 was 2.43±0.46 and 3.18±1.66, and the t max2 were 6.40±1.67 and 5.66±2.02, respectively. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t 1/2 of wogonoside was 4.97±1.68 and 7.71±1.55, the C max1 was 1.39±0.83 and 1.45±0.37, the t max1 was 0.21±0.20 and 0.17±0.01, the C max2 was 1.90±0.55 and 1.42±0.70, and the t max2 was 5.60±1.67 and 5.20±1.79, respectively. A significant difference (p <0.05) was observed for t 1/2, and the elimination of baicalin and wogonoside in Xiaochaihu Tang was increased. [Copyright &y& Elsevier]

Hypothyroidism is known to be closely associated with lipid metabolism. Although our previous serum and urine metabonomics studies have provided some clues about the molecular mechanism of hypothyroidism at the metabolic level, the precise mechanism underlying the pathogenesis of hypothyroidism remains elusive, especially from the aspect of lipid metabolism. In the present study, we applied an ultra high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOF-MS)-based lipidomics method to analyze the global lipid profiles of hypothyroidism in rat cerebellum. Using unsupervised analysis and multivariate statistical analysis, we separated the Sham and hypothyroid groups clearly and screened out 23 potential lipid biomarkers related to hypothyroidism that were primarily involved in sphingolipid metabolism, glycerophospholipid metabolism and β-oxidation of fatty acid. Subsequently, we conducted computational analysis to build and simulate the lipid network of hypothyroidism, knowing that it would be useful to elucidate the pathological mechanism of hypothyroidism. Based on the selected 23 lipid biomarkers, we systematically evaluated the therapeutic effects of Sini decoction ( SND ) and the positive drug T 4 . The results showed that both SND and T 4 can to some extent convert the pathological status of hypothyroidism through different pathways. Overall, this investigation illustrates that lipidomic profiling approach is powerful in giving a complementary view to the pathophysiology of hypothyroidism and offers a valuable tool for systematic study of the therapeutic effects of SND on hypothyroidism at lipid level. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Aconitum carmichaelii (AC) is a well-known herbal medicine for its excellent pharmacological effects and toxicity. The monoester-diterpenoid alkaloids (MDAs), including benzoylmesaconine (BMC), benzoylaconine (BAC) and benzoylhyaconine (BHC), are the main active components in AC. It was found that the diester alkaloids could be transformed into monoester-diterpenoid alkaloids after being decocted. In Chinese pharmacopoeia, the MDAs are also used as phytochemical markers for the quality control of AC. Benzoylmesaconine, benzoylaconine and benzoylhyaconine are representatives of monoester-diterpenoid alkaloids. It was reported that the absolute bioavailability of MDAs was very low but there was toxicity often occurred in AC. Because most of DDAs are transformed into MDAs after decoction, we speculate that some other components may promote the bioavailability of MDAs but result in toxicity by enhancing their absorption. To demonstrate the dynamic changes of MDAs in vivo and reveal the causes of low bioavailability and toxicity, this study will explore the mechanisms of absorption and metabolism of 3 MDAs. Materials and methods: A sensitive, accurate and specific LC-MS method was developed to determine the three MDAs in rat plasma. The pharmacokinetic parameters were estimated after orally administered 3 MDAs to the Male Sprague-Dawley rats, and the metabolism stability was calculated after incubating with rat liver microsomes, finally, the absorption characteristics of the 3 MDAs were investigated using Caco-2 transwell model. Results: It was found that the pharmacokinetic parameters of 3 MDAs were similar, Cmax and Tmax were very small, and t1/2 was large, which indicated 3 MDAs can be absorbed rapidly and is difficult to be metabolized or excreted. However, the low Cmax indicated that the bioavailability of 3 MDAs will be very low and their absorption may be inhibited by some transport proteins. By incubating three MDAs in rat liver microsomes, it was proved that they almost can’t be metabolized in vivo. The Caco-2 transwell experiments reveal that the P-gp inhibits the absorption of MDAs. Conclusions: LC-MS combined with a direct precipitation method for the simultaneous quantification of 3 MDAs in rat plasma has been developed and validated and successfully used in pharmacokinetic study of 3 MDAs. It was proved that the three components almost can’t be metabolized in vivo, and P-gp inhibits the absorption of MDAs. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: The lateral root of Aconitum has been popularly used in traditional Chinese medicine (TMC) known as Fuzi which is beneficial for the treatment of various diseases, such as rheumatism, painful joints, syncope and bronchial asthma. However, it has a potential carditoxicity with a relatively narrow margin of safety. Aim of the study: This paper was designed to explore the mechanisms of Fuzi's toxicity and find out potential tissue-specific biomarkers of toxic effects. Material and methods: A myocardial lipidomics based on ultraperformance lipid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC/Q-TOF MS) was developed to compare three cardiac lipid extraction methods and investigate the changes of lipids in mice heart of three different dosage groups. In addition, we concurrently inspected the biochemical parameters in plasma, observed the histology of the heart and recorded the electrocardiogram (ECG). Results: The cardiotoxicity of Fuzi was dose-dependent, and the high-dose group obviously manifested the heart damage in histology and a certain degree of arrhythmia. Significant changes of 14 lipid metabolites which primarily involved in phospholipid metabolism, sphingolipid metabolism, saturated fatty acid oxidation and unsaturated fatty acid peroxidation were identified and considered as the potential biomarkers of Fuzi toxicity. Conclusion: The lipidomics approach is helpful to search potential tissue-specific biomarkers and understand the underlying mechanisms of Fuzi toxicity on the heart. [Copyright &y& Elsevier]

Saposhnikovia divaricata (SD), a Chinese crude drug, has long been recognized for therapeutic effect to rheumatoid arthritis (RA). At present, the mechanisms of SD treatment in RA have not been fully understood especially on the perspective of metabolomics. Aim of the study : To study the pharmacodynamic effects of Saposhnikovia divaricata decoction on CIA rats, and explore the therapeutic mechanism by metabolomics methods. Wistar rats were randomly divided into normal group, CIA model group, dexamethasone group and SD decoction groups (10 g crude drug/kg, 5 g crude drug/kg and 2.5 g crude drug/kg of SDD). Body weight, arthritis scores, serum cytokine levels and histopathological parameters of rats were assessed. A metabolomics method based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS) was established to collect the metabolic profiles of rats and explore the metabolic changes that occurred after SDD treatment. SDD showed its protective effect on the affected joints, especially in the middle dosage group of SDD. Eighteen and 13 potential biomarkers for the SDD treatment of CIA rats were identified in the plasma and urine, respectively. SDD could regulate the disturbed metabolic pathways including tryptophan metabolism, glycerophospholipid catabolism, primary bile acid biosynthesis and fatty acid metabolism. In summary, SDD treatment could effectively alleviate symptoms of RA and regulate metabolic disorders in CIA rats. Image 1 • Saposhnikovia divaricata decoction could effectively alleviate symptoms of CIA rats. • Differentially biomarkers were identified for the SDD treatment of CIA. • Saposhnikovia divaricata decoction could regulate the disturbed metabolic pathways. [ABSTRACT FROM AUTHOR]