UPLC-ESI-Q-TOF-MS/MS based metabolomics investigation on chemical constituent consistency of Zhenwu Decoction before and after compatibility.

Zhenwu Decoction (ZWD), a classic formula from Zhang Zhongjing's "Treatise on Typhoid Fever" in the Han Dynasty, consists of five traditional Chinese medicines: Aconiti Lateralis Radix Praeparata (ALRP), Paeoniae Radix Alba, Poria Cocos, Ginger, and Rhizoma Atractylodis Macrocephalae. To evaluate the chemical constituent consistency of ZWD before and after compatibility, an ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry was established to comprehensively study the constituents of ZWD. By normalizing the peak area, the pairwise compatibility of ALRP and the other four medicinal herbs, as well as the compatibility of the entire formula were studied, respectively. Multivariate statistical analysis was used to identify the differences. The processed data were analyzed by principal component analysis and supervised orthogonal partial least squared discriminant analysis, and an S-plot was generated to compare the differences in the chemical composition of the two types of decoction samples. The results showed that during the decoction process of ZWD, a total of seven components were recognized as differential compounds before and after compatibility of ZWD, namely 6-gingerol, zingerone, benzoylhypaconine, hypaconitine, benzoylaconine, paeoniflorin and fuziline. The results of this study provide basic data reference for understanding the law of ZWD compatibility and are valuable for the compatibility study of other herbal medicines.

Cucurbit[7]uril as a matrix solid-phase dispersion for the extraction of quaternary ammonium pesticides from vegetables and their determination using HPLC-UV.

Cucurbit[7]uril (Q[7]) was first used as a dispersant sorbent material in a matrix solid-phase dispersion for the simultaneous extraction of four quaternary ammonium pesticides from vegetables before analysis using high-performance liquid chromatography with UV detection. Q[7] exhibited a better selectivity and adsorption capability for these compounds, which is due to its ability to bind selectively organic molecules into its hydrophobic cavity and to form stable host-guest inclusion complexes. Various parameters affecting the extraction were investigated and optimized, such as sorbent/sample mass ratio, grinding time, rinsing and eluting conditions. Under optimized conditions, the proposed method exhibited a linear response in the concentration range of 1-100 µg·kg-1, satisfactory recoveries for eight types of vegetable samples (>70%), and high repeatability (RSD < 9.0%). The limits of quantification were between 0.43 µg·kg-1 and 0.99 µg·kg-1, which is nearly 50 times lower than the maximum residue limits established by the European Council.

Death due to consumption of ibogaine: case report.

Ibogaine is a psychotropic indole alkaloid extracted from the roots of the Tabernanthe iboga shrub from the Apocynaceae family. Depending on the taken dose, it can lead to stimulant effects, euphoria, visual and auditory hallucinations, along with auditory, olfactory, and gustatory synesthesia. In addition to its historical usage in spiritual rituals of African tribes, these days iboga extract presents a prohibited, alternative drug widely used as a part of addiction treatment. Ibogaine used in opioid withdrawal is associated with serious side effects and sudden deaths. Besides its main use as an anti-addiction medication in alternative medicine, in moderate doses (from 100mg to 1g) ibogaine most commonly causes a "trance-like state".In this paper, we report the case of a heroin addict who died suddenly 5-12 hours after oral ingestion of powder labeled Tabernanthe iboga which had been bought online and used in the process of detoxification during an addiction treatment. The man was found dead in a rented apartment, where he was undergoing the addiction treatment.External examination revealed no lesions other than nonspecific injuries on the legs. The autopsy showed congestion of internal organs and pulmonary edema. Histopathological analysis of the heart showed neither macroscopic nor microscopic abnormalities. The concentration of ibogaine was 3.26mg/L. Moreover, systematic toxicological analyses of biological samples showed the presence of morphine and codeine. These data suggest that death, which occurred unnaturally after initiation of the "treatment", was probably the result of the cardiovascular effects caused by the ibogaine powder.The presented case highlights the worldwide problem of various products being widely available over the internet and the danger associated with consumption thereof.

Electrochemical Quantitation of Supramolecular Excipient@Drug Complexation: A General Assay Strategy Based on Competitive Host Binding with Surface-Immobilized Redox Guest.

The macrocyclic cucurbit[7]uril (CB[7]) host has exhibited great application potential as a pharmaceutical excipient due to its versatile abilities to modulate the chemical/physical properties of drug molecules (guests) and to control their in vivo delivery and release (upon complexation). The formation of stable CB[7]@drug complexes is the prerequisite for these promising applications; we report herein a general assay strategy to quantitate the complexation based on competitive binding with surface-immobilized redox guests in conjunction with conventional electrochemical techniques (e.g., cyclic voltammetry). Particularly, by incubating a mixture of CB[7] and a drug molecule with ferrocene (Fc)-terminated self-assembled monolayers (SAMs) on gold, the competitive host@guest binding between the CB[7]@drug complex formed in solution and the CB[7]@Fc complex formed on surface can be quantified with direct cyclic voltammetry measurements. On the basis of the known concentrations of CB[7]/drug and electrochemically determined surface densities of free/complexed Fc groups, the formation constant of CB[7]@drug complex can be determined. With several drug molecules as examples, we have demonstrated the capability of this method for quantitative studies of the formation of supramolecular excipient@drug complexes that are of interest in pharmaceutical and biomedical sciences. More importantly, this work promises a general assay strategy that allows electrochemical quantitation of a wide range of electro-inactive analytes based on the competitive supramolecular host@guest binding at redox-tagged molecular interfaces.

Comparative Transcriptome Analysis Revealed the Tissue-Specific Accumulations of Taxanes among Three Experimental Lines of Taxus yunnanensis.

Taxus yunnanensis (Yew) is known for natural anticancer metabolite paclitaxel (Taxol) and its biosynthesis pathway in yew species still needs to be completely elucidated. In the current study, productions of paclitaxel and 10-DAB III from three different tissues (needle, branch, and root) of T. yunnanensis wild type (WT) and two new cultivars Zhongda-1 (Zd1) and Zhongda-2 (Zd2) were determined, and significant tissue differences in contents of the taxanes were observed among the three experimental lines. The much higher 10-DAB III and lower paclitaxel contents in needle of Zd2 when compared with that of Zd1 indicated the low conversion from 10-DAB III to paclitaxel in the needle of Zd2. In order to uncover the mechanisms of the tissue-specific biosynthesis of the taxanes, transcriptome analysis of cultivar Zd2 was conducted, and the previously reported transcriptome data of Zd1 and WT were used to perform a comparison. The enhancement of TDAT and T10ßH side biosynthetic pathway in roots of Zd2 in early taxane synthesis might lead to the biosynthesis of other toxoids, while the preference of T13αH route in the needle and branch of Zd2 was mainly responsible for the tissue-specific reinforced biosynthesis of 10-DAB III and paclitaxel in Zd2. Different from Zd1, the tissue-specific pattern of paclitaxel biosynthesis genes in Zd2 was similar to WT. However, the lower transcript abundance of final steps genes (TBT, DBAT, BAPT, and DBTNBT) of the paclitaxel biosynthesis pathway in Zd2 than in Zd1 might further promote 10-DAB III accumulation in Zd2.

Genetic Analysis with Random Amplified Polymorphic DNA of the Multiple Enterocin-Producing Enterococcus lactis 4CP3 Strain and Its Efficient Role in the Growth of Listeria monocytogenes in Raw Beef Meat.

In this manuscript, a multiple enterocin-producing Enterococcus lactis strain named 4CP3 was used to control the proliferation of Listeria monocytogenes in refrigerated raw beef meat model. Also, the intraspecific genetic differentiation of 4CP3 strain was assessed by Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) analysis. E. lactis 4CP3 strain was found to produce the enterocins A, B, and P. It displayed activity against L. monocytogenes EGDe 107776 by agar-well diffusion method. The application of E. lactis 4CP3 culture at 107 CFU/g in raw beef meat was evaluated using both ANOVA and ANCOVA linear models in order to examine its effect on the growth of the pathogen L. monocytogenes during refrigerated storage. Hence, a very interesting result in decreasing (P<0.05) and suppressing the growth of L. monocytogenes in refrigerated raw beef meat was shown during 28 days of storage. In conclusion, E. lactis 4CP3 strain might be useful for prevention of the proliferation and survival of L. monocytogenes in raw meat during refrigerated storage.

Application of near-infrared spectroscopy for the rapid quality assessment of Radix Paeoniae Rubra.

Near-infrared (NIR) spectroscopy with multivariate analysis was used to quantify gallic acid, catechin, albiflorin, and paeoniflorin in Radix Paeoniae Rubra, and the feasibility to classify the samples originating from different areas was investigated. A new high-performance liquid chromatography method was developed and validated to analyze gallic acid, catechin, albiflorin, and paeoniflorin in Radix Paeoniae Rubra as the reference. Partial least squares (PLS), principal component regression (PCR), and stepwise multivariate linear regression (SMLR) were performed to calibrate the regression model. Different data pretreatments such as derivatives (1st and 2nd), multiplicative scatter correction, standard normal variate, Savitzky-Golay filter, and Norris derivative filter were applied to remove the systematic errors. The performance of the model was evaluated according to the root mean square of calibration (RMSEC), root mean square error of prediction (RMSEP), root mean square error of cross-validation (RMSECV), and correlation coefficient (r). The results show that compared to PCR and SMLR, PLS had a lower RMSEC, RMSECV, and RMSEP and higher r for all the four analytes. PLS coupled with proper pretreatments showed good performance in both the fitting and predicting results. Furthermore, the original areas of Radix Paeoniae Rubra samples were partly distinguished by principal component analysis. This study shows that NIR with PLS is a reliable, inexpensive, and rapid tool for the quality assessment of Radix Paeoniae Rubra.

Sensitive Immunoassay for Detection and Quantification of the Neurotoxin, Tetramethylenedisulfotetramine.

Tetramethylenedisulfotetramine (TETS, tetramine) is a formerly used and highly neurotoxic rodenticide. Its lethality, recent history of intentional use for mass poisoning, and the absence of a known antidote raise public health concerns. Therefore, rapid, high throughput, and sensitive methods for detection and quantification of TETS are critical. Instrumental analysis method such as GC/MS is sensitive but not rapid or high throughput. Therefore, an immunoassay selective to TETS was developed. The assay shows an IC50 of 4.5 ± 1.2 ng/mL, with a limit of detection of 0.2 ng/mL, comparable to GC/MS. Performance of the immunoassay was demonstrated by a recovery study using known concentrations of TETS spiked into buffer and human and mouse serum matrices giving recoveries in the range of 80-120%. The assay demonstrated good correlation in TETS recovery with established GC/MS analysis. The immunoassay was then used to quantify TETS concentration in the serum of mice exposed to 2× LD50 dose of TETS and to monitor kinetics of TETS clearance from blood over a short period of time. TETS concentration in the serum reached 150 ng/mL without significant change over 4 h post-treatment. Results obtained with the immunoassay had good correlation with GC/MS analysis. Overall, this immunoassay is an important tool to rapidly detect and quantify levels of TETS from biological samples with high sensitivity. The assay can be adapted to multiple formats including field or hospital use.

In vivo detection of cucurbit[6]uril, a hyperpolarized xenon contrast agent for a xenon magnetic resonance imaging biosensor.

The Hyperpolarized gas Chemical Exchange Saturation Transfer (HyperCEST) Magnetic Resonance (MR) technique has the potential to increase the sensitivity of a hyperpolarized xenon-129 MRI contrast agent. Signal enhancement is accomplished by selectively depolarizing the xenon within a cage molecule which, upon exchange, reduces the signal in the dissolved phase pool. Herein we demonstrate the in vivo detection of the cucurbit[6]uril (CB6) contrast agent within the vasculature of a living rat. Our work may be used as a stepping stone towards using the HyperCEST technique as a molecular imaging modality.

Determination of Rodenticide Tetramethylenedisulfotetramine (Tetramine) in Processed Foods by Gas Chromatography-Tandem Mass Spectrometry.

A GC-MS/MS method for determination of the rodenticide tetramethylenedisulfotetramine was developed. Tetramethylenedisulfotetramine was extracted from the sample with ethyl acetate in the presence of anhydrous sodium sulfate. Then, an aliquot of the extract was evaporated under vacuum, followed by acetonitrile/hexane partitioning, and cleanup with a tandem graphitized carbon/primary secondary amine (PSA) column, prior to GC-MS/MS analysis. The recoveries from 10 processed foods, all of which were fortified at 0.1 mg/kg, were in the range of 85-96%, and the relative standard deviations were less than 7%. The proposed method effectively removed co-extracted matrix components, and matrix effects were negligible in the GC-MS/MS analysis. In addition, no interfering peaks were found in the chromatograms of the blank samples at the retention time of tetramethylenedisulfotetramine, indicating that the method is highly selective. Overall results suggest that the proposed method is suitable for determining tetramethylenedisulfotetramine contained in processed foods.

Chemical constituents with anti-allergic activity from the root of Edulis Superba, a horticultural cultivar of Paeonia lactiflora.

The methanolic extract and its subfractions from the dried root of Edulis Superba, a horticultural cultivar of Paeonia lactiflora Pallas, showed potent anti-allergic effect as inhibition of immunoglobulin E (IgE)-mediated degranulation in rat basophil leukemia (RBL)-2H3 cells. Bioassay-guided fractionation led to the isolation of 26 compounds, including a new norneolignan glycoside, paeonibenzofuran (1), together with 25 known ones (2-26). The chemical structure of the new compound was elucidated on the basis of spectroscopic and chemical evidences. Among the isolated compounds, mudanpioside E (5) with paeoniflorin-type skeleton and quercetin (16) showed potent inhibitory activity against a degranulation marker, ß-hexosaminidase release with IC50 values of 40.34 and 25.05 µM, respectively. A primary structure-activity relationship of these components was discussed.

The difference between blood-associated and water-associated herbs of Danggui-Shaoyao San in theory of TCM, based on serum pharmacochemistry.

Danggui-Shaoyao San (DSS) is a famous Chinese formula for activating blood circulation and promoting urination. This study was to investigate the difference of material basis between a blood-associated herbs group and a water-associated herbs group. According to the theory of traditional Chinese medicine, the formula can be divided into a blood-associated herbs group (Angelica sinensis, Paeonia lactiflora and Ligusticum chuanxiong) and a water-associated herbs group (Atractylodes macrocephala, Alisma orientale and Poria cocos). The HPLC fingerprint of the formula was established for quality control. Serum samples from rats, orally administrated DSS, and the decomposed recipes of DSS, were analyzed by HPLC-DAD and the transitional blood components of DSS were identified. Twenty-one common peaks were identified in the fingerprint of DSS. Contents of paeoniflorin, albiflorin, ferulic acid and alisol B 23-acetate in co-decoction were significantly higher than those in individual decoction. Eleven peaks belonged to the blood-associated herbs group (four metabolites and seven prototype components; paeoniflorin and ferulic acid appeared in prototype components), whereas six peaks belonged to the water-associated herbs group (three metabolites and three prototype components). It was concluded that the serum pharmacochemistry is a meaningful approach for clarifying the difference between blood-associated and water-associated herbs in chemical composition.

Simultaneous determination of seven bioactive components in Guizhi Fuling capsule by microwave-assisted extraction combined with ultra performance liquid chromatography tandem mass spectrometry.

A simple, rapid and reliable microwave-assisted extraction (MAE) combined with ultra performance liquid chromatography tandem mass spectrometry method was developed for simultaneous determination of the seven bioactive constituents in Guizhi Fuling capsule (GFC), namely gallic acid, amygdalin, albiflorin, paeoniflorin, paeonol, cinnamic acid and pachymic acid, respectively. The operation of MAE optimised through orthogonal array design experiment was performed at 80°C for 10 min with methanol-water (70:30, v/v) as the extracting solvent. The method was validated including intra- and inter-day precision, repeatability and stability, with relative standard deviation less than 3.9%, 3.3%, 4.4% and 3.1%, respectively. All analytes showed the good linearity (r >0.999), and their average recoveries varied between 98.2% and 101.2%. The results indicated that this method was simple, effective and suitable for the quality control of GFC.

High Affinity Host-Guest FRET Pair for Single-Vesicle Content-Mixing Assay: Observation of Flickering Fusion Events.

Fluorescence-based single-vesicle fusion assays provide a powerful method for studying mechanisms underlying complex biological processes of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated vesicle fusion and neurotransmitter release. A crucial element of these assays is the ability of the fluorescent probe(s) to reliably detect key intermediate events of fusion pore opening and content release/mixing. Here, we report a new, reliable, and efficient single-vesicle content-mixing assay using a high affinity, fluorophore tagged host-guest pair, cucurbit[7]uril-Cy3 and adamantane-Cy5 as a fluorescence resonance energy transfer (FRET) pair. The power of these probes is demonstrated by the first successful observation of flickering dynamics of the fusion pore by in vitro assay using neuronal SNARE-reconstituted vesicles.

Changes in gene transcription and taxane production in elicited cell cultures of Taxus × media and Taxus globosa.

The response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), supplied to the culture medium either separately or together, was studied. Two-stage Taxus globosa and Taxus media cell cultures were established and the elicitors were added at the beginning of the second stage. Growth, taxane production, and the expression of known taxol biosynthetic genes, including the recently characterized CoA ligase gene, were studied. Although CORO reduced the growth capacity of both cell lines, CD apparently counteracted this negative effect. Taxane production was significantly enhanced by the simultaneous addition of CD and CORO to the medium. The total taxane production in the T. media cell line was more than double that of T. globosa, but in the latter more than 90% of the taxanes produced were excreted to the medium. Individual taxane patterns also differed: at the height of production, the main taxanes in T. globosa cultures were cephalomannine and 10-deacetyltaxol, and in T. media, taxol and baccatin III. The low transcript levels of taxane biosynthetic genes found in T. globosa cells mirrored the lower taxane production in these cultures, while a high expression was strongly correlated with a high taxane production in T. media.

Genetic and chemical characterization of white and red peony root derived from Paeonia lactiflora.

Two kinds of peony roots--white peony root (WPR) and red peony root (RPR)--are used for different remedies in traditional Chinese medicine; however, most of them are derived from the same botanical origin, Paeonia lactiflora. The difference between WPR and RPR has been debated for a long time. This study attempted to clarify the genetic and chemical characteristics of WPR and RPR in order to provide a scientific dataset for their identification and effective use. The nucleotide sequence of nrDNA internal transcribed spacer (ITS) and the contents of 8 main bioactive constituents were analyzed from specimens of P. lactiflora, P. veitchii and two related species as well as crude drug samples of WPR, RPR and peony root produced in Japan. Of the samples derived from P. lactiflora, the WPR produced in the southern parts of China and the RPR produced in the northern parts of China were clearly divided into two subgroups within the P. lactiflora group based on similarity of the ITS sequences. The nucleotides at positions 69, 458 and 523 upstream of the ITS sequence served as molecular markers to discriminate between WPR and RPR. Quantitative analysis indicated that the RPR samples obviously contained a higher content of paeoniflorin and paeonol, but a lower content of albiflorin than the WPR produced in the southern parts of China and peony root produced in Japan. The WPR available from Chinese markets was usually processed by sulfur fumigation, which resulted in an extremely low content of paeoniflorin. This study indicated that WPR and RPR were not only geographically isolated, but also genetically and chemically separated. The ITS sequence provided a genetic index for their identification.

Genetic and component content differentiation between wild and cultivated populations of Paeonia lactiflora and related species used as Chishao and Baishao in China.

One hundred and forty-four samples of Chishao and Baishao, which represented six species of Paeonia L. were evaluated for their genetic variation, genetic differentiation and phylogenetic relationship, based on the nuclear ribosomal DNA internal transcribed spacer (ITS) region. Samples from representative of the population were then used to do a cultivation comparison experiment, and then to identify the contents of the active ingredients. The results showed there were differences in the haplotype distribution and frequency between populations of Chishao and Baishao. An analysis of molecular variance (AMOVA) indicated statistically significant (p<0.001) genetic differentiation between the populations of wild and cultivated Paeonia lactiflora PALL. The albiflorin content between Chishao and Baishao was also significantly different (p<0.05). All the results clearly illustrate that currently cultivated P. lactiflora cannot be used as a substitute for Chishao.

Simultaneous determination of 11 major components in Palmul-tang by HPLC-DAD and LC-MS-MS.

Palmul-tang, a traditional herbal medicine, is composed of eight herbs (Ginseng radix, Glycyrrhizae radix, Hoelen, Atractylodis rhizoma, Angelicae gigantis radix, Cnidii rhizoma, Paeoniae radix and Rehmanniae radix) and exhibits various bioactivities, including antiallergic and antitumor effects. In this study, an effective, reliable and accurate high-performance liquid chromatography method has been developed for the simultaneous determination of 11 marker components in Palmul-tang: hydroxymethylfurfural, albiflorin, paeoniflorin, ferulic acid, nodakenin, ginsenoside Rg1, decursinol, glycyrrhizin, 6-gingerol, ginsenoside Rg3 and decursin. All calibration curves of the 11 components indicated excellent linearity (correlation coefficient > 0.9997) within the test range. The limits of detection and quantification of each component were in the ranges of 0.08-1.03 and 0.23-3.11 µg/mL, respectively. The intra-day and inter-day relative standard deviation values were within 1.65 and 2.71%, respectively. The mean recovery values were 94.49 to 101.10%. The established method was successfully applied to the simultaneous determination of 11 major components in 12 commercial samples of Palmul-tang. The developed analytical method is simple and suitable for the quality control of Palmul-tang.

[Simultaneous determination of paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol in zhengtian pills by HPLC].

To simultaneously determine paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol in Zhengtian pills. In the test, Insertil ODS-C18 column (4.6 mm x 250 mm, 5 microm) was adopted, with acetonitrile-0.05% phosphoric acid solution as the mobile phase for gradient elution. The flow rate was 1.0 mL x min(-1), the column temperature was 30 degrees C and the detection wavelength was 230 nm. According to the results of the test, paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol showed good linear relations between peak areas and sample sizes in 11.37-170.5, 2.188-32.82, 2.896-43.44, and 3.000-45.00 mg x L(-1) (r = 0.999 9, 0.999 9, 1.000 0, 1.000 0), respectively. The average recoveries (n = 6) were 102.3% (RSD 1.2%), 99.71% (RSD 1.9%), 101.2% (RSD 1.2%), and 99.40% (RSD 2.4%), respectively. The above four components were determined in five batches of samples by using the established method, with satisfactory results. The method was so simple, accurate and highly reproducible that it could be used for quality control of the four components in Zhengtian pills.

Fatal case of a 27-year-old male after taking iboga in withdrawal treatment: GC-MS/MS determination of ibogaine and ibogamine in iboga roots and postmortem biological material.

We report the case of a man who died twelve hours after ingesting powdered iboga root, commonly taken for its stimulant and hallucinogenic properties. Ibogaine and ibogamine were quantified in the powder ingested and the victim's body fluids by GC-MS/MS after liquid-liquid extraction (Toxi-tubes A(®)). The concentrations of ibogaine measured in the blood samples taken at the scene and in the peripheral blood, urine, and gastric fluid samples taken during the autopsy were 0.65, 1.27, 1.7, and 53.5 µg/mL, while the iboga content in the powder was 7.2%. Moreover, systematic toxicological analyses of biological samples showed the presence of diazepam and methadone in therapeutic concentrations. Death was attributed to the ingestion of a substantial quantity of iboga in the context of simultaneous methadone and diazepam consumption.