Polyamidoamine (PAMAM) dendrimers as synthetic gene vectors are efficient gene delivery systems. In this study, a kind of α-cyclodextrin-PAMAM conjugates polymer (CyD-G1) was synthesized as a gene delivery vector. Based on ¹H NMR detectation, about 6.4 PAMAM-G1 molecules was grafted onto an α-CD core. Agarose gel electrophoresis revealed that CyD-G1 could efficiently bind with DNA to condense them into nano-scale particles, which showed a similar binding capacity of PEI-25K. Besides, it could protect DNA from DNase I degradation in a low N/P ratio. When N/P ratio in the CyD-G1/DNA polyplex was 40, the average particle size of CyD-G1/DNA polyplex was about 120 nm, and zeta potential was +21 mV. This polyplex could maintain its particle size in serum-containing solution within 360 min. In comparison with PEI-25K carrier, CyD-G1 showed low cytotoxicity in various cell lines. Cell transfection results showed that CyD-G1 efficiently delivered DNA into cells at N/P=80 compared with Lipofectamine 2000 and PEI-25K.Unlike Lipofectamine 2000 and PEI-25K, in serum-containing test condition, CyD-G1/DNA polyplex could maintain the transgene activities. The results of confocal laser scanning microscopy indicated that most DNA entered into cell nuclei within 4 h, and this phenomenon was consistent with the results calculated by flow cytometry. Taken together, CyD-G1 showed good transgene activities and the gene delivery vector could be used not only in vitro but also in vivo.

The major non-P450 enzymes involved in the oxidative metabolism of drugs are:the flavincontaining monooxygenase (FMO), the monoamine oxidase (MAO), the aldehyde oxidase (AO), the xanthine oxidase (XO), the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). In recent years, the role of non-P450 enzymes in drug oxidative metabolism has garnered increasing attention. However, the contribution of non-P450 enzymes to the drug oxidative metabolism is possibly underestimated in many cases, as most metabolism studies in drug discovery and lead optimization are conducted using in vitro test systems related to P450 enzymes. In this article, these non-P450 enzymes in terms of catalyzed reaction types, common substrates, gene polymorphism and drug interaction are reviewed, and the in vitro models and factors for non-P450-mediated oxidative metabolism are summarized. Similar to P450 enzymes, non-P450 enzymes can directly catalyze the oxidation of drugs, yielding therapeutically active metabolites or toxic metabolites. These enzymes can also oxidize the toxic metabolites, generated from P450-catalyzed reaction, to nontoxic metabolites. In general, most non-P450 enzymes (such as FMO and MAO) appear to be much less inducible than P450 enzymes.

As important constituents of the first-line of host defense barrier, intestinal cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp) play important roles in disease pathogenesis as well as drug absorption and exposure. Clinical reports and experimental data revealed diminished intestinal CYP3A and P-gp expression accompanying with gut dysbiosis in inflammatory bowel disease. Yet whether gut dysbiosis is associated with the down-regulation of CYP3A and P-gp and the underlying mechanisms are unclear. In this study, daily administration of fresh feces from normal rats and rats with ulcerative colitis (UC) induced by dextran sulfate sodium to normal rats resulted in alterations of gut bacterial compositions. Intestinal CYP3A2 and P-gp were significantly down-regulated in rats receiving UC feces. Outer-membrane vesicles (OMVs) are nano-scale special buds of the outer membrane which are produced by Gram-negative bacteria and mediate diverse functions including interactions within bacterial communities and communications with host. Expressions of CYP3A4 and P-gp mRNA were diminished in human epithelial colorectal adenocarcinoma cells (Caco-2) treated by OMVs from all different groups with OMVs from UC rats or rats receiving UC feces showing more significant effects.Moreover, the OMVs fractions within 30 000-50 000 Daltons from both normal and UC rats elicited more effects than fractions of other molecular weights. Treatment of Caco-2 cells with toll like receptor 4 (TLR4) inhibitor resatorvid (TAK-242) or TLR4 silence RNA (siRNA) blocked CYP3A4 and P-gp down-regulation induced by bacterial OMVs. Taken together, we proved in this study that gut microbiota can down-regulate intestinal CYP3A and P-gp partially through producing OMVs to activate the TLR4 signaling pathway.

This study was conducted to investigate the inhibitory effect and the molecular mechanism of deoxyschizandrin on the activity of NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome. Bone marrow-derived macrophages were used to study the effects of deoxyschizandrin on inflammasome activation using inflammasome inducers (ATP and nigericin). Cytotoxic effect was evaluated with CCK-8. The expression of IL-1β, caspase-1 in the supernatant and the expression of pro-caspase-1, pro-IL-1 β, ASC, NLRP3 in cell was detected by Western blot for the inhibitory effect of deoxyschizandrin (25, 50, 100 and 200 μmol·L^-1) on the activity of NLRP3 inflammasome. Immunofluorescence was applied to investigate NF-κB (p65) transportation to the nucleus. The results of CCK-8 showed that the optimum concentration of deoxyschizandrin was 6.25-400 μmol·L^-1. Deoxyschizandrin (25, 50, 100, and 200 μmol·L^-1) could inhibit the activation of NLRP3 inflammasome caused by nigericin and ATP, and inhibit the secretion of IL-1 β, which was associated with inhibiting the cleavage of pro-caspase-1. The results of immunofluorescence and Western blot also suggest that the inhibitory activity of deoxyschizandrin on NLRP3 inflammasome was not dependent on NF-κB pathway and protein expression of NLRP3, ASC, pro-caspase-1 and pro-IL-1 β mediated by NF-κB. Our results confirmed that deoxyschizandrin could suppress the cleavage of pro-caspase-1 and inhibit the activity of NLRP3 inflammasome at 25-200 μmol·L^-1 to reduce the inflammation response.

This study was designed to clarify the chemical constituents in Yuanhu Zhitong prescription (YHZT), a rapid high performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry (HPLC-QTOF/MS) method was established. Based on the high resolution MS spectra data, fragmentation ion information, reference standards data and literature reports, 51 peaks including 28 alkaloid compounds and 23 coumarin compounds were identified. The chemical constituents in YHZT were rapidly, accurately, systematically analyzed. The results lay a foundation for the quality control of effective compounds of YHZT.

Carboxylesterase 1 (CE1) is an important serine hydrolase in mammals, which involved in the hydrolysis of a variety of compounds (endogenous substrates like cholesterol and xenobiotic compounds like ester-contain drugs and pesticides). This study aimed to design and develop the fluorescent probe substrates for human carboxylesterase 1 (hCE1), on the basis of the structural features of hCE1 preferred substrates. Four carboxylic esters deriving from BODIPY-8-carboxylic acid were designed and synthesized. After then, reaction phenotyping assays and chemical inhibition assays were used to evaluate the selectivity of these four ester derivatives towards hCE1. Our results clearly demonstrated that the substrate specificity of these ester substrates towards hCE1 would be improved with the decrease of the alcohol group on BODIPY-8-carboxylesters, while BODIPY-8-carboxylesters with small alcohol groups including methyl (BCM) and ethyl (BCE) esters could serve as the ideal probe substrates for hCE1. Given that BCM exhibit rapid hydrolytic rate in hCE1, we further investigate the enzymatic kinetics of this fluorescent probe substrate in both human liver microsomes (HLM) and recombinant hCE1, as well as to explore its potential application in high-throughput screening of hCE1 inhibitors by using HLM as enzyme source. The results showed that the kinetic behaviors and the affinity of BCM in HLM is much closed to those in recombinant hCE1, implying that hCE1 played the key roles in BCM hydrolysis in HLM. Furthermore, the inhibition study demonstrated that BCM could be used for rapid screening and characterization of hCE1 inhibitors, by using HLM to replace recombinant hCE1 as enzyme source.

Cytochrome P4502J2 (CYP2J2) is widely distributed in various human tissues and takes a part in the metabolism of endogenous compounds and drugs. CYP2J2 can convert arachidonic acid (AA) to expoxyeicosatrienoic acids (EETs), which have various biological effects, implying the important role of CYP2J2 in the regulation of cardiovascular system and promotion of tumor progression and metastasis. Additionally, CYP2J2 plays an indispensable role in the intestinal metabolism of various drugs, such as astemizole, terfenadine and ebastine. In this review, the metabolic function, characteristic of catalysis and tissue distribution of CYP2J2 are discussed with the latest literatures both in China and abroad. The state-of-the-art methods for characterization of CYP2J2 and current trend of substrate discovery as well as its relationship with disease are highlighted. This review gives in-depth understanding of the function of CYP2J2 and its role in disease advance. The information of ligand (substrate and inhibitor) will provide the theoretical guidance and reference to the development of novel drugs for CYP2J2.

Bupleuri Radix is one of the most frequently used herbal medicines in China with a 2000-year medicinal history. However, the use of Bupleuri Radix is very confused. Twenty-five species and eight varieties of Bupleurum have been used as Bupleuri Radix in different regions of China. It is very difficult to identify these Bupleurum species using traditional morphological method. In order to establish a fast and effective method to identify these Bupleurum species, we collected 168 Bupleurum medicinal plants from 14 populations of 9 provinces, and amplified their ITS sequences. 168 ITS sequences with a full length of 600-606 bp were obtained. DNAMAN analyzing results showed that 86 variable sites were present in these sequences and 19 haplotypes (TH1-TH19) were determined. After calculating K2P distance and analyzing an NJ tree, we established a molecular identification method based on ITS sequence. Using this method,52 samples of Bupleuri Radix were identified successfully. Furthermore, we tested saikosaponin a, c, d contents in these Bupleuri Radix by HPLC and analyzed the results by ANOVA and LSD T test to evaluate the quality of Bupleuri Radix. This method is significant for effective identification of Bupleurum medicinal plants, and quality control of Bupleuri Radix in the market.

Tropane alkaloids are anticholinergic drugs widely used clinically. Biosynthesis of tropane alkaloids in planta involves a step of transamination of phenylalanine. Based on the sequenced transcriptomes of lateral roots and leaves of Hyoscyamus niger, we found three annotated aromatic amino acid aminotransferases, which were respectively named HnArAT1, HnArAT2 and HnArAT3. Sequence analysis showed that HnArAT3 had highest similarity with the reported Atropa belladonna AbArAT4, which was involved in tropane alkaloid (TA) to provide the precursor of the phenyllactic acid moiety. Tissue expression pattern analysis indicated that HnArAT3 was specifically expressed in lateral roots, where is the organ synthesizing tropane alkaloids. Then, method of virus induced gene silencing (VIGS) was used to characterize the function of HnArAT3 in H. niger. Gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had lower expression levels of HnArAT3 than the non-transgenic control, and HPLC analysis of alkaloids demonstrated significant decrease in the contents of hyoscyamine, anisodamine and scopolamine in planta. These results suggested that HnArAT3 was involved in the phenyllactic acid branch of TA biosynthetic pathway. Molecular cloning and functional identification of HnArAT3 laid the foundation for further understanding of TA biosynthesis and metabolic regulation, and also provided a new candidate gene for engineering biosynthetic pathway of tropane alkaloids.

Thiochromanones and 1,3,4-thiadazoles as heterocyclic compounds have broad biological activities. In order to find novel compounds with antifungal bioactivity, substituted thiophenol and maleic anhydride were used to synthesize the intermediate 4-oxothiochromane-2-carboxylic acid. It was reacted with 2-amino-1,3,4-thiadiazol to get fourteen target compounds containing 1,3,4-thiadazole moiety. The structures of the obtained compounds were confirmed by ¹H NMR, ¹³C NMR and HR-MS. All compounds were investigated for antifungal activity via microdilution broth method. The results showed that the target compounds 3a and 3c to Epidermophyton floccosum and Mucor racemosus exhibited better antifungal activity than the positive control fluconazole, in which the minimum inhibition concentration can reach 8 μg·mL^-1 and 16 μg·mL^-1. Compound 3e showed significant inhibitory activity to Helminthosporium maydis, Sclerotinia sclerotiorum and Botrytis cinerea compared with that of the positive control carbendazim. Compound 3b exhibited inhibitory activity to Helminthosporium maydis better than the positive control carbendazim.