Pharmacokinetics of sanguinarine, chelerythrine, and their metabolites in broiler chickens following oral and intravenous administration.

Sanguinarine (SA) and chelerythrine (CHE) are the main active components of the phytogenic livestock feed additive, Sangrovit®. However, little information is available on the pharmacokinetics of Sangrovit® in poultry. The goal of this work was to study the pharmacokinetics of SA, CHE, and their metabolites, dihydrosanguinarine (DHSA) and dihydrochelerythrine (DHCHE), in 10 healthy female broiler chickens following oral (p.o.) administration of Sangrovit® and intravenous (i.v.) administration of a mixture of SA and CHE. The plasma samples were processed using two different simple protein precipitation methods because the parent drugs and metabolites are stable under different pH conditions. The absorption and metabolism of SA following p.o. administration were fast, with half-life (t1/2 ) values of 1.05 ± 0.18 hr and 0.83 ± 0.10 hr for SA and DHSA, respectively. The maximum concentration (Cmax ) of DHSA (2.49 ± 1.4 µg/L) was higher that of SA (1.89 ± 0.8 µg/L). The area under the concentration vs. time curve (AUC) values for SA and DHSA were 9.92 ± 5.4 and 6.08 ± 3.49 ng/ml hr, respectively. Following i.v. administration, the clearance (CL) of SA was 6.79 ± 0.63 (L·h-1 ·kg-1 ) with a t1/2 of 0.34 ± 0.13 hr. The AUC values for DHSA and DHCHE were 7.48 ± 1.05 and 0.52 ± 0.09 (ng/ml hr), respectively. These data suggested that Sangrovit® had low absorption and bioavailability in broiler chickens. The work reported here provides useful information on the pharmacokinetic behavior of Sangrovit® after p.o. and i.v. administration in broiler chickens, which is important for the evaluation of its use in poultry.

Identification of gelsemine metabolites in rat liver S9 by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

RATIONALE: Gelsemine has been extensively studied because of its anti-tumor, immunomodulatory, insecticidal itching and other significant effects. However, limited information on the pharmacokinetics and metabolism of gelsemine has been reported. Therefore, the purpose of the present study was to investigate the in vitro metabolism of gelsemine in rat liver S9 by using rapid and accurate high-performance liquid chromatography/ quadrupole-time-of-flight mass spectrometry (HPLC/QqTOF-MS). METHODS: The incubation mixture was processed with 15% trichloroacetic acid. Multiple scans of gelsemine metabolites and accurate mass measurements were automatically performed simultaneously through data-dependent acquisition in only 30 min. The structural elucidations of these metabolites were performed by comparing their changes in accurate molecular masses and product ions with those of the parent drug. RESULTS: Five metabolites of gelsemine were identified in rat liver S9. Of these, four metabolites of gelsemine were identified for the first time. The present results showed that the metabolic pathways of gelsemine are oxidation, demethylation, and dehydrogenation in rat liver S9. CONCLUSIONS: In this study, metabolites of gelsemine in liver S9 were identified and elucidated firstly using the HPLC/QqTOF-MS method. The proposed metabolic pathways of gelsemine in liver S9 will provide a basis for further studies of the in vivo metabolism of gelsemine in animals and humans.

Identification of allocryptopine and protopine metabolites in rat liver S9 by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

RATIONALE: Allocryptopine (AL) and protopine (PR) have been extensively studied because of their anti-parasitic, anti-arrhythmic, anti-thrombotic, anti-inflammatory and anti-bacterial activity. However, limited information on the pharmacokinetics and metabolism of AL and PR has been reported. Therefore, the purpose of the present study was to investigate the in vitro metabolism of AL and PR in rat liver S9 using a rapid and accurate high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC/QqTOFMS) method. METHODS: The incubation mixture was processed with 15% trichloroacetic acid (TCA). Multiple scans of AL and PR metabolites and accurate mass measurements were automatically performed simultaneously through data-dependent acquisition in only a 30-min analysis. The structural elucidations of these metabolites were performed by comparing their changes in accurate molecular masses and product ions with those of the precursor ion or metabolite. RESULTS: Eight and five metabolites of AL and PR were identified in rat liver S9, respectively. Among these metabolites, seven and two metabolites of AL and PR were identified in the first time, respectively. The demethylenation of the 2,3-methylenedioxy, the demethylation of the 9,10-vicinal methoxyl group and the 2,3-methylenedioxy group were the main metabolic pathways of AL and PR in liver S9, respectively. In addition, the cleavage of the methylenedioxy group of the drugs and subsequent methylation or O-demethylation were also the common metabolic pathways of drugs in liver S9. In addition, the hydroxylation reaction was also the metabolic pathway of AL. CONCLUSIONS: This was the first investigation of in vitro metabolism of AL and PR in rat liver S9. The detailed structural elucidations of AL and PR metabolites were performed using a rapid and accurate HPLC/QqTOFMS method. The metabolic pathways of AL and PR in rat were tentatively proposed based on these characterized metabolites and early reports. Copyright © 2016 John Wiley & Sons, Ltd.

Structural speculation and identification of alkaloids in Macleaya cordata fruits by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry combined with a screening procedure.

RATIONALE: Alkaloids with significant therapeutic effects are the main active constituents of Macleaya cordata, which is a perennial herb plant in the Papaveraceae family. A systematic and novel method for speculating and identifying the structures of alkaloids in M. cordata fruits by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC/Q-TOF-MS) with a screening procedure was reported. METHODS: Investigation of mass spectral fragmentation of alkaloids was carried out based on the tandem mass spectrometry (MS/MS) data analyses of eight reference substances. The skeletons of alkaloids were determined by their ultraviolet spectra (UV) and MS/MS data. The substituent groups of the alkaloids were acquired through a screening procedure developed in our laboratory and MS/MS data. The substituent linkage sites were deduced by MS/MS fragmentation behavior, as well as biosynthetic pathways of related alkaloids. RESULTS: The structures of 21 alkaloids were speculated in this study, 10 of which were reported for the first time in M. cordata. Furthermore, benzyltetrahydroisoquinoline and N-methyltetrahydroprotoberberine-type alkaloids were discovered, which indirectly proved that the biosynthetic pathways of benzophenanthridine alkaloids reported in Eschscholtzia california existed in M. cordata as well. CONCLUSIONS: HPLC/Q-TOF-MS combined with a screening procedure was a systematic and reliable method for speculating and elucidating the structures of alkaloids. This study might be useful for the identification of other compounds in herbal medicines.

Research on the Efficacy of Ganpu Vine Tea in Inhibiting Uric Acid Production.

Ganpu vine tea is a new type of health care citrus fruit tea made from citrus shell, Pu-er tea, and vine tea baked as raw materials. In this study, the in vitro uric acid synthase inhibition system and hyperuric acid cell model were constructed to appraise the uric acid lowering efficacy of Ganpu vine tea, traditional Ganpu tea, and vine tea. Results showed that in the uric acid synthase inhibition system, the aqueous extract can inhibite the puric metabolically related enzymes, such as adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and xanthine oxidase (XOD). The ability of the aqueous extract to inhibit the above enzyme was as follows: vine tea > Ganpu vine tea > Ganpu tea; all teas had a strong effect on XOD inhibition. The hyperuric acid cell model test showed that the aqueous extract inhibited uric acid production through accumulating inosine and hypoxanthine and hindering xanthine synthesis. The uric acid reductive ability was as follows: Vine tea > Ganpu vine tea > Ganpu tea. The inhibition of enzymes related to uric acid synthesis and the inhibition of uric acid production were significantly enhanced through adding vine tea to Ganpu tea. It also shows that flavonoids are the main factor driving this ability because they are the main active ingredients in these botanical drinks.

Molecular Characterization and Phylogenetic Analysis of Spirometra Tapeworms from Snakes in Hunan Province.

Sparganosis is a neglected zoonotic parasitic disease that poses huge threats to humans worldwide. Snakes play an important role in sparganosis transmission because they are the most common second intermediate hosts for Spirometra parasites. However, the population genetics of Spirometra isolates from snakes is currently not well studied in China. The present study was performed to explore the molecular characteristics and phylogenetic analysis of Spirometra tapeworms from different species of snakes in Hunan Province. This study obtained 49 Spirometra isolates from 15 geographical areas in Hunan Province, Central China. Subsequently, the 18S and 28S ribosomal DNA (rDNA) fragments were amplified from the isolated parasites, and their sequences were analyzed to assess their genetic diversity. Phylogenetic analyses were performed using the maximum likelihood algorithm. The results showed that sequence variations among these isolates were 0-2.3% and 0-0.1% for 18S and 28S rDNA, respectively. The phylogenetic analysis showed that all Spirometra isolates from Hunan Province were clustered into the same branch with Spirometra erinaceieuropaei isolated from other areas (China, Vietnam, Australia). Moreover, the phylogenetic trees revealed that Spirometra is closely related to Adenocephalus, Pyramicocephalus, Ligula, Dibothriocephalus, Schistocephalus, and Diphyllobothrium. The Spirometra isolates of different hosts/regions in Hunan Province are not host segregated or geographically isolated, and support for the taxonomic status of Spirometra tapeworms in China has been added. These results provide reference values for future accurate identification and taxonomic status of Spirometra tapeworms in China.

Exploring the interaction between Cry1Ac protein and Zn2+, Cd2+ metal ions by fluorescence quenching and molecular docking approaches.

Bacillus Thuringiensis (Bt) protein has a strong ability to complex with metal ions, which may increase the transport of metal ions in the soil multi-media system. In this study, the interactions between Cry1Ac protein and metal ions (Zn2+ and Cd2+) were investigated through spectroscopies and molecular docking methods. The spectra results showed that both Zn2+ and Cd2+ quenched the fluorescence intensity of Cry1Ac protein through the static quenching. The binding constants with 4-5 orders of magnitude also indicated the interactions between the ions and the Cry1Ac protein. The thermodynamic analysis showed that hydrogen bonds and van der Waals forces were predominant during the processes. In terms of the Förster non-radiation energy transfer theory, the binding distances between metal ions and Cry1Ac protein were approximately 0.21-0.24 nm, indicating the existence of a non-radiative energy transfer between them. Furthermore, molecular docking revealed that the metal ions participated in ligand binding with the Cry1Ac at the locations Asp569, Thr560, Asn564 and Gln566. The present work provided reasonable models helping us further understand the transport effect of heavy metals in the presence of Cry1Ac. The results could provide mechanistic insights into the nature of metal ions-Cry1Ac interactions and offer important information on the toxicity risk of metal ions-Cry1Ac binding interactions.

Metabolism and Tissue Distribution of Chelerythrine and Effects of Macleaya Cordata Extracts on Liver NAD(P)H Quinone Oxidoreductase.

Background: Macleaya cordata (Willd.) (Papaveraceae) is listed as a feed additive in animal production by the European Food Authority. Methods: The metabolites of chelerythrine in rats were measured in vitro and in vivo by rapid and accurate high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC/QqTOF-MS). The structures of CHE metabolites were elucidated by comparing their changes in accurate molecular masses and fragment ions with those of parent ion or metabolite. The metabolic enzymes that were involved in chelerythrine reduction were investigated using an inhibition method. The tissue distribution of chelerythrine and the effects on NQO1 following intragastric administration with M. cordata extracts in rats were examined. Results: A total of twelve metabolites of chelerythrine were characterized by this approach in rat liver S9 and in vivo. The reduction of the iminium bond of chelerythrine and subsequent O-demethylation was the main metabolic pathway of chelerythrine in rat liver S9 while the reduction of the iminium bond of chelerythrine was the main metabolic pathway of chelerythrine in rats in vivo. After the rats were given intragastric administration, the low concentration residues of sanguinarine and chelerythrine in different rat tissues were found at 48 h after the last dose, suggesting that both compounds could be widely distributed in tissues. The results also indicated that XO, NQO1, NQO2, and carbonyl reductase are involved in chelerythrine reduction. Macleaya cordata extracts treated female and male rats, respectively, showed different responses, inhibiting NQO1 activity in males, but inducing NQO1 activity in females. Chelerythrine had a weak impact on NQO1 activity, but sanguinarine inhibited NQO1 activity Conclusion: Through studying the effects of cytosolic reductase inhibitors on chelerythrine reduction and the impact of chelerythrine and sanguinarine on the activity of NQO1 in vitro and in vivo, we clarified the potential drug interaction of Macleaya cordata extract in clinical application, so as to provide theoretical guidance for clinically safe medication. In addition, it provided a reference basis for the metabolic mechanism of chelerythrinein rats.

MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes.

MicroRNAs (miRNAs/miRs) are considered to serve essential roles in podocyte apoptosis, and to be critical in the development of diabetic nephropathy (DN). Activation of the Notch signaling pathway has been demonstrated to serve an important role in DN development; however, its regulatory mechanisms are not fully understood. The present study used a high glucose (HG)-induced in vitro apoptosis model using mouse podocytes. Expression levels of miR-145-5p and its target, Notch1, and other key factors involved in the apoptosis signaling pathway were detected and measured by reverse transcription-quantitative PCR and western blotting. A luciferase reporter assay was performed to elucidate the miRNA-target interactions. The functions of miR-145-5p in apoptosis were detected using flow cytometry and TUNEL staining. The present study demonstrated that in HG conditions, miR-145-5p overexpression inhibited Notch1, Notch intracellular domain, Hes1 and Hey1 expression at the mRNA and protein levels. Notch1 was identified as a direct target of miR-145-5p. Furthermore, cleaved caspase-3, Bcl-2 and Bax levels were reduced significantly by miR-145-5p overexpression. These results indicate that miR-145-5p overexpression inhibited the Notch signaling pathway and podocyte lesions induced by HG. In conclusion, the results of the present study suggested that miR-145-5p may be a regulator of DN. Additionally, miR-145-5p inhibited HG-induced apoptosis by directly targeting Notch1 and dysregulating apoptotic factors, including cleaved caspase-3, Bcl-2 and Bax. The results of the present study provided evidence that miR-145-5p may offer a novel approach for the treatment of DN.

Over-expression of Bcl2-associated athanogene 2 in oral cancer promotes cellular proliferation and is associated with poor prognosis.

OBJECTIVE: The aim of the present study was to state the role of BAG2 in oral squamous cell carcinomas (OSCC). DESIGN: Expression data of BAG2 in OSCC tissues were extracted from Oncomine and TCGA database. Expression levels of BAG2 mRNA and protein were examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot assay. The Kaplan-Meier method was conducted to evaluate the overall survival of OSCC patients. Small interfering RNAs (siRNAs) strategy was used to confirm the effect of BAG2 expression on proliferative, invasive, migrated capacities of OSCC cells by Cell Counting kit-8 (CCK-8), colon formation assay, wound healing and transwell assay. RESULTS: Our results showed that BAG2 expression was up-regulated in oral squamous cell carcinomas tissues. Compared with OSCC patients with low BAG2 expression, poorer overall survival rate was found in OSCC patients with high BAG2 expression. Furthermore, proliferation, invasion and migration of HO-1-N-1 cells were significantly inhibited because of the knockdown of BAG2. Transfection of si-BAG2 has no impacts on proliferation in HNOEC cells. Inhibition of BAG2 downregulated the expression of relevant proteins, such as proliferating cell nuclear antigen (PCNA), c-Myc, matrix metalloproteinase-2 (MMP-2) and Vimentin. Additionally, the expression levels of the important protein phosphorylation (p-ERK1/2 and p-MEK) in mitogen-activated protein kinase (MAPK) pathway were reduced in HO-1-N-1 cells transfected with si-BAG2. CONCLUSIONS: High-regulated BAG2 is related to poor prognosis and could promote proliferation, invasion and migration of OSCC cells by activating the MAPK signaling pathway. Thus, BAG2 may be a potential target for OSCC therapy.

Proteomics analysis of faecal proteins in the tick Haemaphysalis flava.

BACKGROUND: Ticks and tick-borne diseases are of major public health concern. Currently, development of vaccines against ticks is considered crucial for their control. A critical step in this process is the screening of viable antigens. Faeces are byproducts of digestion and blood meal utilization, and partly reflect the vitality and vector potential of ticks. However, an integrated analysis of proteins in tick faeces is lacking. The present study explored the protein components in the faeces of the tick Haemaphysalis flava, by liquid chromatography-tandem mass spectrometry (LC/MS-MS) to identify potential protein antigens for vaccine development against ticks. METHODS: Faeces from adult H. flava engorged females were collected. Proteins were extracted from faeces, and the trypsin-digested peptides were analyzed by LC/MS-MS. High confidence proteins were identified based on unique peptides revealed by MS. Potential faecal protein genes, as well as their sources, were also characterized by searching previous transcriptome datasets from the salivary glands and midgut of H. flava. RESULTS: In total, 21 were recognized with confidence. Amongst these, 18 were of likely tick origin, while three proteins (serum albumin, haemoglobin α and ß subunits) were likely from hosts. Seventeen unigenes corresponding to these proteins were retrieved by searching our previous H. flava salivary glands and midgut transcriptomic datasets. Some proteins were reported to prevent blood clotting, play a role in immunity and antibiosis, and formation of musculature. The functions of the remaining proteins are unknown. CONCLUSIONS: Identifying antigens for tick vaccine development is feasible by analyzing the faecal proteome as well as the transcriptomes of salivary glands and midguts. The vast number of proteins detected in tick faeces highlights the complexity of blood digestion in ticks, a field that needs more investigation.

[Appropriate application of statistical methods in physiological research].

OBJECTIVE: To offer a series of efficient methods to physiologists in appropriate selection, and application of statistical techniques. METHODS: We bring about two questions as follows:What's the role of statistics in the process of a physiological research? How to make sure the results produced in a physiological research can be repeatable in practice in the long run. From the answers to these two questions, we highlight the importance of the discipline of statistics to research work, explain why it is difficult, how to choose a suitable statistical method in a specific situation, and offer the critical methods to use statistics accurately and appropriately. RESULTS: We abstract three core sections from the discipline of statistics:how to make the design of a study impeccable; How to strictly follow the protocol of a study, and How to draw conclusions well reasoned and strongly supported by evidence. By elaborating these sections, it is feasible to correctly use statistical methods for data analysis and results interpretation. CONCLUSIONS: In physiological research, conclusion can stand with time and repeatable in practice only when researchers strictly and rigorously follow the rule of scientific research.

[How to scientifically estimate sample size in physiological research].

OBJECTIVE: To bring about physiological researchers' attention of the importance of sample size estimation. METHODS: The significance as well as the current problems of sample size estimation were illustrated and the commonly-used sample size estimation methods were introduced. RESULTS: The basic concepts and necessary premises of sample size estimation were stated. The estimation processes and results under two different circumstances were elaborated in detail via examples. CONCLUSIONS: To attain the proper estimated sample sizes, the computation must satisfy the necessary premises which included the appropriate statistical analysis methods to be used.

Systematic identification of alkaloids in Macleaya microcarpa fruits by liquid chromatography tandem mass spectrometry combined with the isoquinoline alkaloids biosynthetic pathway.

Alkaloids in Macleaya microcarpa were characterized systematically by combining liquid chromatography tandem mass spectrometry (LC-MS/MS) with the biosynthetic pathway of isoquinoline alkaloids. The mass spectral fragmentation behaviors of 16 references belonging to eight types of alkaloids that exist in the biosynthetic pathway of isoquinoline were investigated in detail. The benzyltetrahydroisoquinoline and aporphine alkaloids were distinguished by characteristic losses of the NHR1R2 (R1 and R2 represent the substituent groups of the nitrogen atom) radical and the fragment ions below m/z 200. Tetrahydroprotoberberine, N-methyltetrahydroberberine and protopine alkaloids were differentiated by the retro-Diels-Alder (RDA) reaction, α-cleavage and the [M-H2O](+) and [M-CH4](+) ions. Discrimination of protoberberine, benzophenanthridine and dihydrobenzophenanthridine-type alkaloids can be realized through the characteristic [fragment ion-2H](+), [M-H2O](+), [M-CH4](+), [M+H-CH3CH2CH2OH](+) and [M+H-CH3COCH3](+) ions. Forty-one alkaloids, including one benzyltetrahydroisoquinoline, one aporphine, nine protopines, seven protoberberines, one tetrahydroprotoberberine, three N-methyltetrahydroprotoberberines, five benzophenanthridines and fourteen dihydrobenzophenanthridines, were separated and identified simultaneously. Thirty-three of these were reported for the first time in M. microcarpa. The benzyltetrahydroisoquinoline, aporphine, tetrahydroprotoberberine and N-methyltetrahydroprotoberberine-type alkaloids have not been reported previously in M. microcarpa. This method can be applied to the analysis of herbal medicines that possess the biosynthetic pathway of isoquinoline alkaloids.

DEPD: a novel database for differentially expressed proteins.

SUMMARY: The Differentially Expressed Protein Database was designed to store the output of comparative proteomics studies and provides a publicly available query and analysis platform for data mining. The database contains information about more than 3000 differentially expressed proteins (DEPs) manually extracted from the published literature, including relevant biological, experimental and methodological elements. Tools for visualization and functional analysis of DEPs are provided via a user-friendly webinterface. AVAILABILITY: http://protchem.hunnu.edu.cn/depd/.