Study on network pharmacology of Ginkgo biloba extract against ischaemic stroke mechanism and establishment of UPLC-MS/MS methods for simultaneous determination of 19 main active components.

INTRODUCTION: Ginkgo biloba extract (GBE) is an effective substance from traditional Chinese medicine (TCM) G. biloba for treating ischaemic stroke (IS). However, its active ingredients and mechanism of action remain unclear. OBJECTIVES: This study aimed to reveal the potential active component group and possible anti-IS mechanism of GBE. MATERIALS AND METHODS: The network pharmacology method was used to reveal the possible anti-IS mechanism of these active ingredients in GBE. An ultra-high-performance liquid chromatography triple quadrupole electrospray tandem mass spectrometry (UPLC-MS/MS) method was established for the simultaneous detection of the active ingredients of GBE. RESULTS: The active components of GBE anti-IS were screened by literature integration. Network pharmacology results showed that the anti-IS effect of GBE is achieved through key active components such as protocatechuic acid, bilobalide, ginkgolide A, and so on. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the possible anti-IS mechanism of GBE is regulating the PI3K-Akt signalling pathway and other signal pathways closely related to inflammatory response and apoptosis regulation combined with AKT1, MAPK, TNF, ALB, CASP3, and other protein targets. Nineteen main constituents in seven batches of GBE were successfully analysed using the established UPLC-MS/MS method, and the results showed that the content of protocatechuic acid, gallic acid, ginkgolide A, and so forth was relatively high, which was consistent with network pharmacology results, indicating that these ingredients may be the key active anti-IS ingredients of GBE. CONCLUSION: This study revealed the key active components and the anti-IS mechanism of GBE. It also provided a simple and sensitive method for the quality control of related preparations.

Molecular Determinants Elucidate the Selectivity in Abscisic Acid Receptor and HAB1 Protein Interactions.

The abscisic acid (ABA), as a pivotal plant hormone, plays a key role in controlling the life cycle and adapting to the environmental stresses. The receptors of ABA are the Pyrabactin resistance/Pyrabactin resistance-like/regulatory component of ABA receptors (PYR/PYL/RCAR, PYLs for simplicity), which regulate the protein phosphatase 2Cs (PP2Cs) in the signal pathway. As an important ABA-mimicking ligand, Pyrabactin shows the activation function to parts of members of PYLs, such as PYR1 and PYL1. Due to the antagonism of Pyrabactin to PYL2, it was used as a probe to discover a part of ABA receptors. Since then, many researchers have been trying to find out the determinants of the selective regulation of PYLs and PP2Cs interaction. However, the roles of residues on the selective regulation of PYR1/PYL2 and PP2Cs interaction induced by Pyrabactin are still ambiguous. This research investigated the selective activation mechanism of Pyrabactin through the sequence alignment, molecular docking, molecular dynamics simulation, and binding free energy calculation. Furthermore, the electrostatic and hydrophobic interaction differences induced by Pyrabactin and agonists were compared. The results indicate that Leu137/Val114, Ser85/Ser89, and Gly86/Gly90 from the pocket and gate of PYR1/PYL2 are the vital residues for the selective activation of Pyrabactin. Meanwhile, the electrostatic interaction between PP2Cs and PYLs complexed with agonists was improved. This mechanism provides strong support for the design of selective agonists and antagonists.

In vivo analysis of two new fungicides in mung bean sprouts by solid phase microextraction-gas chromatography-mass spectrometry.

A novel solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) method was developed to quantify two new fungicide residuals (Y13149, Y12196) in mung bean sprouts. With a stable and biocompatible elcetrospinning nanofiber (polystyrene/graphene@silica, PS/G@SiO2) as coating, the SPME fiber was directly inserted into the stem of mung bean sprout to in-situ in-vivo sampling and extraction, followed by GC-MS analysis. Under the optimal conditions, satisfactory average recoveries of 99% and 72% were obtained for Y13149 and Y12196 with the relative standard deviations (RSDs) under 16.3%, indicating good precision and anti-matrix ability of the method. The result also exhibited low detection limit (0.06-0.08 µg·L-1) and wide liner range (0.3-100 µg·L-1) with the correlation coefficients (R2) of 0.9989. The established method was applied successfully to trace the accumulation and distribution of fungicides in mung bean sprouts, and it provides a simple, rapid and reliable quantitative method for food analysis.

Computational Discovery of Potent and Bioselective Protoporphyrinogen IX Oxidase Inhibitor via Fragment Deconstruction Analysis.

Tuning the binding selectivity through appropriate ways is a primary goal in the design and optimization of a lead toward agrochemical discovery. However, how to achieve rational design of selectivity is still a big challenge. Herein, we developed a novel computational fragment generation and coupling (CFGC) strategy that led to a series of highly potent and bioselective inhibitors targeting protoporphyrinogen IX oxidase. This enzyme plays a vital role in heme and chlorophyll biosynthesis, which has been proven to be associated with many drugs and agrochemicals. However, existing agrochemicals are nonbioselective, resulting in a great threat to nontargeted organisms. To the best of our knowledge, this is the first bioselective inhibitor targeting the tetrapyrrole biosynthesis pathway. In addition, the candidate showed excellent in vivo bioactivity and much better safety toward humans.

Enhanced butanol production in a microbial electrolysis cell by Clostridium beijerinckii IB4.

Reducing power such as NADH is an essential factor for acetone/butanol/ethanol (ABE) fermentation using Clostridium spp. The objective of this study was to increase available NADH in Clostridium beijerinckii IB4 by a microbial electrolysis cell (MEC) with an electron carrier to enhance butanol production. First of all, a MEC was performed without electron carrier to study the function of cathodic potential applying. Then, various electron carriers were tested, and neutral red (NR)-amended cultures showed an increase of butanol concentration. Optimal NR concentration (0.1 mM) was used to add in a MEC. Electricity stimulated the cell growth obviously and dramatically diminished the fermentation time from 40 to 28 h. NR and electrically reduced NR improved the final butanol concentration and inhibited the acetone generation. In the MEC with NR, the butanol concentration, yield, proportion and productivity were increased by 12.2, 17.4, 7.2 and 60.3 %, respectively. To further understand the mechanisms of NR, cathodic potential applying and electrically reduced NR, NADH and NAD(+) levels, ATP levels and hydrogen production were determined. NR and electrically reduced NR also improved ATP levels and the ratio of NADH/NAD(+), whereas they decreased hydrogen production. Thus, the MEC is an efficient method for enhancing the butanol production.

[Pollution characteristics and accumulation of antibiotics in typical protected vegetable soils].

The occurrence and distribution of 14 selected antibiotics in soils from 20 protected vegetable fields in Shandong province were investigated by ultrasonic extraction and UPLC-MS/MS. The results showed that antibiotics were detected in all the soil samples, the dominant antibiotics were tetracycline, oxytetracycline, chlortetracycline and doxycycline with 100% detection rate, the concentration of which in soil ranged from 2.11-139.16, 6.06-332.02, 1.82-391.31, 2.20-248.56 microg x kg(-1), respectively. The total concentration of four compounds sigma (TCs) ranged from 26.79-1010.11 microg x kg(-1), with an average of 274 microg x kg(-1). All the sulfonamides (SAs), quinolones (QNs) and macrolides antibiotics (MACs) were also detected, except for chloramphenicoles. The total concentration of QNs [sigma (QNs)] in soils ranged from 0-1017.06 microg x kg(-1) and the average concentration was 73.05 microg x kg(-1) with detection rate of 85%, and the individual concentrations of SAs and MACs were quite low in soils. The results also showed that the distibution and concentration of antibiotics in soils grown different vegetables were quite different. Notably, the individual concentrations of QNs (NFC, OFC) were 373.73 microg x kg(-1) and 643.34 microg x kg(-1), respectively, which far exceeded the trigger value of the ecological risk (100 microg x kg(-1)). Thus, more attention should be paid to antibiotics pollution in protected vegetable soils.

[Screening of differentially expressed genes during adipocyte differentiation by suppression subtractive hybridization technique].

OBJECTIVE: To screening differentially expressed genes related to adipocyte differentiation. METHODS: Total RNA extracted from the preadipocyte cell line SW872 was taken as the Driver and the total RNA from the differentiated adipocytes SW872 as the Tester. Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes. The products of SSH were inserted into pGM-T vector to establish the subtractive library. The library was amplified through E.coli transformation and positive clones of the transformants were screened. Positive clones were sequenced. Nucleic acid similarity was subsequently analyzed by comparing with the data from GenBank. RESULTS: There were 135 white clones in the cDNA library, 64 positive clones were chosen randomly and sequenced and similarity search revealed 34 genes which expressed differentially in adipocyte differentiation. CONCLUSION: The subtracted cDNA library for differentially expressed in adipocyte differentiation has been successfully constructed and the interesting candidate genes related to adipocyte differentiation have been identified.

[Effect of early high fat diet on pancreatic ß cellularity and insulin sensibility in young rats].

OBJECTIVE: To study the effects of early high fat diet on sugar metaboliam, insulin sensibility and pancreatic ß cellularity in young rats. METHODS: Sixty male weaned young rats were randomly fed with high fat diet (high fat group) and normal diet (control group). The body weight, viscus fattiness and fasting plasma glucose (FPG) were measured after 3, 6 and 9 weeks. Serum insulin level was measured with radioimmunoassay. The ultrastructure of pancreas was observed under an electricmicroscope. RESULTS: The high fat group had significantly higher body weight and visceral fat weight than the control group after 3 weeks. There were no significant differences in the FPG level between the two groups at all time points. The levels of fasting insulin and HOMAIR in the high fat group were significantly higher than those in the control group after 3, 6 and 9 weeks (P<0.01). Dilation of rough endoplasmic reticulum and mild swelling of mitochondria of islet ß-cells were observed in the high fat group after 6 weeks. CONCLUSIONS: Early high fat diet may induce a reduction in insulin sensitivity and produce insulin resistance in young rats. Endoplasmic reticulum expansion in ß-cells may be an early sign of ß-cell damage due to obesity.

[Dynamic expression of matrix metalloproteinases 2 and 8 in rat cervix].

OBJECTIVE: To elucidate the relationship between collagen degradation and cervical ripening by detecting dynamic expressions of matrix metalloproteinases 2 (MMP-2) and 8 (MMP-8) in rat cervix. METHOD: SPF rats were divided into 5 groups (n=6), namely non-pregnancy estrus interval group, gestational days 10, 16, and 19 groups, and immediately postpartum group. The wet weight of the cervix was measured and HE staining was used to display the general structure of the cervix. VG staining was applied to visualize the collagen fibers and muscular fibers. Immunohistochemistry was performed to observe the expressions of MMP-2 and MMP-8 in the cervix. RESULTS: HE staining showed that the rat uterine cervix consisted mainly of fibroblasts and fibrous connective tissues. A small quantity of neutrophils could be seen in the cervix stroma of the rats immediately after immediately parturition, but not at the other time points. The wet weight of the antepartum cervix had increased, and a more obvious increase was seen in the wet weight of the cervix immediately after parturition. The collagen fibers of the cervix consisted of collagen fibers and smooth muscle fibers, and their proportions showed no significant variation at the time points around the parturition. Immediately after parturition, the collagen fibers and muscular fibers in the cervix became loosened as compared with that before parturition. MMP-2 expression was found in the cervical stroma but not in the squamous epithelium in nonpregnancy, term pregnancy, and immediately after parturition; the smooth muscle cells, vascular wall, and stromal fibroblasts showed positive expression of MMP-2. Enhanced intensity of MMP-2 staining was seen in term pregnancy and postpartum group in comparison with that in the other groups. MMP-8 expression was observed in the cervix of rats immediately after parturition, with scattered neutrophils positive for MMP-8 spotted in the stroma of the ripened cervix. MMP-8 expression was not detected in the other groups. CONCLUSION: Ripened cervical fibrous tissue becomes loose and broken, and cervical ripening is accompanied by infiltration of neutrophils from exogenous vessels. These changes are particularly evident after parturition. MMP-2 and MMP-8 cooperate to degrade the cervical fibers, leading to cervical softening and expansion.

[Association between marital quality and hypertensive disorder in pregnancy].

OBJECTIVE: To investigate the sociopsychological basis of hypertensive disorder in pregnancy (HDP) and explore a new pathway for etiological study of HDP. METHODS: A prospective investigation was conducted in 1154 women in second trimester pregnancy and 9 factors were surveyed using Olson marital quality questionnaire (ENRIC). The discrepancy between the norms and factor scores of ENRIC was analyzed, and the scores of ENRIC were compared between normal gravidas and patients with HDP. The correlation between ENRIC scores and the severity of the condition was also evaluated. RESULTS: The score of the 1124 gravidas for marital satisfaction was significantly higher than the norm (P<0.05), but the scores for relationship with relatives and sexual life were significantly lower (P<0.05). The other 6 factors had similar scores with the norms (P>0.05). Patients with HDP had significantly lower scores for 7 factors than the normal gravidas (P<0.05), but had comparable scores for financial arrangement and sexual life (P>0.05). The severity of HDP was not found to associate with variation of the scores for the 9 factors (P>0.05). CONCLUSIONS: Marital quality is an important social and psychological basis of HDP, and this study provides some evidence for the social and psychological investigation of the etiology of HDP.

[Characteristics of uterine contraction and stages of labor under continuous epidural block anesthesia].

OBJECTIVE: To observe the characteristics of uterine contraction and stages of labor during delivery under continuous epidural block anesthesia. METHODS: Totaling 213 parturients in spontaneous labor under epidural block anesthesia with dilated cervical orifice of 3 cm were monitored for the contraction cycle, duration, intensity and curve types of uterine contraction, and recordings were made for 30 min before and 30, 60 and 120 min after the anesthesia took effect, respectively. The duration of the active phase in the first, second and third stages of labor was compared between 421 cases with anesthesia and 237 without anesthesia. RESULTS: Significant difference was noted in the objective indexes of uterine contraction recorded after anesthesia had taken effect (P<0.05) in comparison with those before anesthesia, suggesting significantly attenuated uterine contraction after anesthesia, whereas these indexes underwent no significant further variation as compared between different time points after anesthesia (P>0.05). The average active phase in the first stage was significantly shorter in anesthesia group than that in the control group (P<0.05), but the average duration of the second and third stages of labor differed little between the two groups with appropriate use of oxytocin under strict monitoring (P>0.05). The rates of obstetric forceps utilization and use of oxytocin were higher in anesthesia group than in the control group (P<0.05). CONCLUSION: Epidural block anesthesia produces certain influences on uterine contraction and stages of labor during delivery, for which appropriate treatment measures may prove beneficial.

[Effect and risk analysis of misoprostol in stimulating cervical maturity for post-term pregnancy].

OBJECTIVE: To evaluate the effect and risk of misoprostol for stimulating cervical maturity in women with post-term pregnancy negative for insulin-like growth factor binding protein-1 (IGFBP-1) in cervical secretion with modified Bishop score less than 3. METHODS: Seventy-one women with post-term pregnancy randomized into misoprostol group (n=37) and control group (n=34) received misoprostol placement at the posterior vaginal fornix and routine intravenous oxytocin infusion, respectively, to stimulate cervical maturity. Failure to respond to the treatment within the initial 24 h necessitated a repeated administration for no more than 3 times in all. Modified Bishop score was recorded and fetal heart monitored once every 24 h, and IGFBP-1 in the cervical secretion was detected at 24 and 48 h after drug administration. RESULTS: The misoprostol group showed better effect of cervical maturity stimulation than the control group (P<0.001), and the positivity rates of IGFBP-1 24 and 48 h after drug administration were significantly higher than that of the control group (P<0.01 and 0.001). The number of cases with indication for cesarean section was significant higher in the control group (P<0.001). There were no significant differences in postpartum hemorrhage, excessive uterine contraction, incidence of fecal contamination of the amniotic fluid or Apgar score of the newborn between the two groups (P>0.05). CONCLUSIONS: Misoprostol is safe and effective for stimulating cervical maturity in women with post-term pregnancy who have modified Bishop score lower than 3 and are negative for IGPBF-1 in cervical secretion. Oxytocin is not advised for use in such gravida for stimulating cervical maturity. IGFBP-1 in cervical secretion may serve as an important index for evaluating the cervical maturity.

High-density lipoprotein as a potential carrier for delivery of a lipophilic antitumoral drug into hepatoma cells.

AIM: To investigate the possibility of recombinant high-density lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells. METHODS: Recombinant complex of HDL and aclacinomycin (rHDL-ACM) was prepared by cosonication of apoproteins from HDL (Apo HDL) and ACM as well as phosphatidylcholine. Characteristics of the rHDL-ACM were elucidated by electrophoretic mobility, including the size of particles, morphology and entrapment efficiency. Binding activity of rHDL-ACM to human hepatoma cells was determined by competition assay in the presence of excess native HDL. The cytotoxicity of rHDL-ACM was assessed by MTT method. RESULTS: The density range of rHDL-ACM was 1.063-1.210 g/mL, and the same as that of native HDL. The purity of all rHDL-ACM preparations was more than 92%. Encapsulated efficiencies of rHDL-ACM were more than 90%. rHDL-ACM particles were typical sphere model of lipoproteins and heterogeneous in particle size. The average diameter was 31.26+/-5.62 nm by measure of 110 rHDL-ACM particles in the range of diameter of lipoproteins. rHDL-ACM could bind on SMMC-7721 cells, and such binding could be competed against in the presence of excess native HDL. rHDL-ACM had same binding capacity as native HDL. The cellular uptake of rHDL-ACM by SMMC-7721 hepatoma cells was significantly higher than that of free ACM at the concentration range of 0.5-10 microg/mL (P<0.01). Cytotoxicity of rHDL-ACM to SMMC-7721 cells was significantly higher than that of free ACM at concentration range of less than 5 microg/mL (P<0.01) and IC50 of rHDL-ACM was lower than IC50 of free ACM (1.68 nmol/L vs 3 nmol/L). Compared to L02 hepatocytes, a normal liver cell line, the cellular uptake of rHDL-ACM by SMMC-7721 cells was significantly higher (P<0.01) and in a dose-dependent manner at the concentration range of 0.5-10 microg/mL. Cytotoxicity of the rHDL-ACM to SMMC-7721 cells was significantly higher than that to L02 cells at concentration range of 1-7.5 microg/mL (P<0.01). IC50 for SMMC-7721 cells (1.68 nmol/L) was lower than that for L02 cells (5.68 nmol/L), showing a preferential cytotoxicity of rHDL-ACM for SMMC-7721 cells. CONCLUSION: rHDL-ACM complex keeps the basic physical and biological binding properties of native HDL and shows a preferential cytotoxicity for SMMC-7721 hepatoma to normal L02 hepatocytes. HDL is a potential carrier for delivering lipophilic antitumoral drug to hepatoma cells.