NB-LRR genes: characteristics in three Solanum species and transcriptional response to Ralstonia solanacearum in tomato.

MAIN CONCLUSION: NB-LRR genes in the three Solanum species showed specific constitution characteristics and evolved multiple clusters and duplicates. Some genes could respond to biotic stresses such as tomato bacterial wilt. Nucleotide-binding and leucine-rich repeat (NB-LRR, NLR) is a largest resistance gene family in plants, which plays a key role in response to biotic stresses. In this study, NB-LRR genes in cultivated tomato Solanum lycopersicum (Sl) and its wild relatives S. pennellii (Spe) and S. pimpinellifolium (Spi) were analyzed using bioinformatics approaches. In total, 238, 202 and 217 NB-LRR genes of 8 different types were found in Sl, Spe and Spi, respectively. The three species showed similar genomic characteristics. The NB-LRR genes were mainly distributed on chromosomes 4, 5 and 11 and located at the distal zones, forming multiple clusters and tandem duplicates. A large number of homologs appeared through gene expansion, with most Ka/Ks values being less than 1, indicating that purifying selection had occurred in evolution. These genes were mainly expressed in root and could respond to different biotic stresses. RT-qPCR analysis revealed that SlNLR genes could respond to tomato bacterial wilt, with SlNLR1 probably involved in the resistance response, whereas others being the opposite. The transcription factors (TFs) and interaction proteins that regulate target genes were mainly Dof, NAC and MYB families and kinases. The results provide a basis for the isolation and application of related genes in plant disease resistance breeding.

[Metabonomic study on the anti-liver injury effect of Si-Ni-San on rats by using UPLC-MS/MS].

A UPLC-MS/MS method based on metabonomic skills was developed to study the serum metabolic changes of rats after acute liver injury induced by CCl4 and to evaluate the action mechanism of Si-Ni-San. The integrated data were exported for principal components analysis (PCA) by using SIMCA-P software, in order to find the potential biomarkers. It showed that clear separation of healthy control group, model group, silymarin group, Si-Ni-San group was achieved by using the PCA method. Nine significantly changed metabolites were identified as potential biomarkers of acute liver injury. Compared with the health control group, the model group rats showed higher levels of phenylalanine, tryptophan and GCDCA together with lower levels of LPC 16 : 0, LPC 18 : 0, LPC 18 : 1, LPC 16 : 1, LPC 20 : 4 and LPC 22 : 6. These changes of serum metabolites suggested that the disorders of amino acid metabolism, lipid metabolism, bile acid biosynthesis and anti-oxidative damage were related to acute liver injury induced by CCl4. Si-Ni-San might have the anti-liver injury effect on all these four metabolic pathways.

[Effect of phenformin hydrochloride on pharmacokinetics of puerarin in rats].

OBJECTIVE: To study the effect of phenformin hydrochloride that may be illegally added in traditional Chinese medicine preparations on the pharmacokinetics of puerarin in rats. METHOD: Rats were randomly divided into the single pueraria group and the phenformin hydrochloride combined with pueraria group. After oral administration in the two groups, their bloods were sampled at different time points to determine the drug concentration of puerarin in rat blood and calculate pharmacokinetic parameters. RESULT: After oral administration with pueraria extracts and phenformin hydrochloride combined with pueraria extracts, the two groups showed main pharmacokinetic parameters as follows: Cmax were (2.39 +/- 1.01), (1.03 +/- 0.35) mg x L(-1), respectively; Tmax were (0.50 +/- 0.09), (1.5 +/- 0.5) h, respectively; Ke were (0.153 +/- 0.028), (0.172 +/- 0.042) h(-1), respectively; t(1/2) were (4.65 +/- 0.86), (4.20 +/- 0.81) h, respectively; AUC(0-t), were (5.73 +/- 2.60), (5.45 +/- 1.81) mg x h x L(-1), respectively; AUC(0-infinity) were (6.72 +/- 2.89), (6.26 +/- 1.88) mg x h x L(-1), respectively. Compared with the single puerarin group, the Cmax was significantly decreased (P < 0.05) and the Tmax was markedly longer (P < 0.01) than the hydrochloride combined with pueraria group. CONCLUSION: Phenformin hydrochloride can slow down the absorption process of puerarin and change the pharmacokinetic process of puerarin to some extent.

[LC-MS/MS method for quantification and pharmacokinetic study of gabapentin in human plasma].

A sensitive, rapid and specific liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for quantification of gabapentin in human plasma has been developed. After a single plasma protein precipitation with methanol, gabapentin and metformin (internal standard) were chromatographed on a Inertsil ODS-3 column (50 mm x 2.1 mm ID, 3 microm) with mobile phase consisting of methanol-0.2% formic acid aqueous solution (80:20, v/v) at a flow-rate of 0.2 mL x min(-1). Electrospray ionization (ESI) source was applied and operated in the positive ion mode. Multiple reaction monitoring (MRM) mode with the transitions of m/z 172 --> m/z 154 and m/z 130 --> m/z 71 were used to quantify gabapentin and metformin, respectively. The run time was 2.2 min. The linear calibration curve was obtained in the concentration range of 40.8-8.16x10(3) ng x mL(-1). The lower limit of quantification was 40.8 ng x mL(-1). The intra- and inter-day precision (RSD) was less than 12%, and the accuracy (RE) was within +/-6.4% calculated from quality control (QC) samples. The method was used to determine the concentration of gabapentin in human plasma after a single oral administration of 600 mg gabapentin capsule to 20 healthy male Chinese volunteers. The method was proved to be selective, sensitive, rapid and suitable for pharmacokinetic study of gabapentin in human plasma.

Pharmacokinetics and safety evaluation in healthy Chinese volunteers of alkaloids from leaf of Alstonia scholaris: A multiple doses phase I clinical trial.

BACKGROUND: Alstonia scholaris (Apocynaceae) was reported to be a rich source of indole alkaloids, which exhibited remarkably bioactivities. The leaf of A. scholaris has been used in 'dai' ethno-medicine for treatment of respiratory diseases, and the defined indole alkaloids from leaf of A. scholaris has been registered as investigational new botanical drug (No. 2011L01436) and was approved for phase I/II clinical trials by China Food and Drug Administration (CFDA). PURPOSE: The aim of the trial is to evaluate the safety and explore the relationship of dosing frequency and pharmacokinetics after oral administration of capsule of alkaloids from leaf of A. scholaris (CALAS) at different doses. METHODS: In this randomized, open-labelled, single-center clinical trial, the safety and pharmacokinetics of CALAS were assessed in eligible healthy Chinese volunteers after oral administration of different doses. Each volunteer (n = 10 per group) received single dose of CALAS from 20 mg, 40 mg, 80 mg to 120 mg orally. The pharmacokinetics of CALAS was investigated in healthy Chinese subjects' plasma by a fully-validated LC-MS/MS method. Safety was assessed biochemically and clinically throughout the study, and drug re-excitation research was conducted to verify the correlation between investigational product and minor adverse events. The trial was registered on August 26, 2015 (http://www.chictr.org.cn/showproj.aspx?proj=11736), number ChiCTR-IPR-15006976. RESULTS: 40 subjects completed the study, and as a result, vallesamine had the highest concentration in plasma of healthy volunteers, and the AUC exposure level in each compounds in turn is vallesamine > scholaricine > 19-epischolaricine > picrinine. For the safety evaluation of CALAS, two cases of minor adverse events were observed during the trial, but the drug re-excitation research indicated that these two adverse events were related to the individual's physiological variation. CONCLUSION: Pharmacokinetic characteristics of each ingredient showed different patterns. 19-epischolaricine, vallesamine and picrinine were match to the linear pharmacokinetic characteristics, but scholaricine conformed to the characteristics of nonlinear pharmacokinetics. The CALAS was safe in healthy subjects under the current dose regimen.

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.

Simultaneous quantification of 10 saponins in Chinese Shizhu Panax by UPLC-ESI-MS.

A simple and rapid method was established and validated for the simultaneous quantification of 10 saponins, namely ginsenosides-Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, Re, Rf and Notoginsenside R1, in Chinese Shizhu Panax by ultra performance liquid chromatography coupled with an electrospray mass spectrometry (UPLC-ESI-MS). In addition, the contents of the analytes in different parts of Chinese Shizhu Panax were also analysed. The results showed that the concentration of saponins had a reference to the different parts of Chinese Shizhu Panax. The established method could be used as a new analytical approach for assessment of the quantity of Chinese Shizhu Panax.

Analysis of main constituents and mechanisms underlying antidepressant-like effects of Xiaochaihutang in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaochaihutang (XCHT), a famous Chinese herbal formula which consists of seven Chinese herbs, has been used clinically in depressive disorders in China. Our previous studies have demonstrated that XCHT improved depressive-like behavior in several animal models of depression. However, therapeutic basis of XCHT on depression are challenging, due to the complex active constituents of XCHT and the unclear pharmacological mechanism of action. MATERIALS AND METHODS: To provide further insights into therapeutic basis of XCHT, the core in compatibility of XCHT on antidepressant therapy was assessed by the method of orthogonal array design. The comparative evaluations on antidepressant effects of XCHT and its core in compatibility were executed by tail suspension test (TST), forced swim test (FST), novelty suppressed feeding test (NSFT), reserpine-induced hypothermia and palpebral ptosis. Moreover, the potential mechanism was explored by investigating levels of monoamine neurotransmitters in hypothalamus and striatum and neurogenesis in hippocampus. Chemical profile of active constituents in plasma after oral administration of the core in compatibility of XCHT was revealed by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: The results of orthogonal array design experiment showed that Huangqin (Radix scutellariae), Renshen (Ginseng) and Gancao (Radix glycyrrhizae), defined as HRG, might be the core in compatibility of XCHT on antidepressant therapy. In accordance with XCHT, oral administration of HRG for 15 days significantly reduced immobility duration in TST and FST without affecting locomotor activity. Both HRG and XCHT increased immobility latency in FST, decreased the latency in NSFT, reversed reserpine-induced hypothermia and palpebral ptosis. Moreover, both HRG and XCHT significantly increased levels of 5-HT and DA in hypothalamus. In addition, HRG could remarkably increase Ki-67 and doublecortin (DCX) positive cells in hippocampus. A total 25 active constituents in plasma, including 14 prototype components and 11 metabolites, were identified by UPLC-MS/MS after oral administration of HRG. CONCLUSION: The present results reveal that HRG is supposed to be the core in compatibility of XCHT on antidepressant therapy. In accordance with XCHT, HRG exerts significant antidepressant-like effects, which are likely attributed to regulating serotonergic and dopaminergic systems and increasing hippocampal neurogenesis. The constituents identified in plasma after oral administration of HRG may be the potential active ingredients for the treatment of depression.

[HPLC determination of two flavonoid compounds in Psoralea corylifolia].

OBJECTIVE: To determine two flavonoid compounds in Psoralea corylifolia L. (PC) simultaneously with HPLC method. METHOD: Bavachin and corylin isolated from PC and purified in our laboratory were used as the reference compounds. The HPLC separation was carried out on an Techsphere ODS column using mobile phase consisting of a mixture of methanol and 20 mmol.L-1 ammonium acetate buffer pH 4.0 (67:33), and the UV detection wavelength was 240 nm. RESULT: Simultaneous determination of bavachin and corylin was achieved. The linear range was 1.25-20 micrograms.mL-1 for both bavachin and corylin. The average recovery of bavachin and corylin was 94.9% and 96.2%, and RSD was 3.1% and 3.6% respectively. CONCLUSION: This is the first report on simultaneous determination of bavachin and corylin in PC with satisfactory accuracy and reproducibility.

Xiaochaihutang prevents depressive-like behaviour in rodents by enhancing the serotonergic system.

OBJECTIVES: Xiaochaihutang (XCHT) has been used in China for thousands of years to treat 'Shaoyang syndrome', which involves depressive-like symptoms. However, no studies were conducted to demonstrate its antidepressant effect and mechanism. This study was designed to confirm the antidepressant effect of XCHT and explore its mechanism using the pharmacological methods. METHODS: Ultra-HPLC and mass spectrometry was used to identify the chemical constituents of XCHT. Forced swimming test (FST) and tail suspension test (TST) were used to determine the antidepressant-like activity of XCHT in mice and rats. The possible mechanism of XCHT was elucidated by the reserpine-induced hypothermia and 5-hydroxytryptophan (5-HTP)-induced head-twitch in mice. The levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were measured in prefrontal cortex and hippocampus tissue of both mice and rats. Moreover, the extracellular 5-HT in rat hippocampus was assessed by using microdialysis coupled to HPLC with electrochemical detection. KEY FINDINGS: Forty-four components were detected in XCHT. XCHT significantly reduced immobility time in the TST and the FST, antagonized reserpine-induced depressive-like behaviours, increased 5-HTP-induced head-twitches, elevated 5-HT and 5-HIAA levels, and increased 5-HT turnover at doses that did not affect general activity. CONCLUSIONS: These data demonstrate that XCHT has therapeutic effects in animal models of depression by enhancing the serotoninergic system in the prefrontal cortex and hippocampus.