[Mechanism of Qiwei Guibao Granules in treatment of premature ovarian failure based on proteomics].

In this study, the underlying mechanism of Qiwei Guibao Granules(QWGB) in the treatment of premature ovarian fai-lure(POF) was explored by the proteomics technique. Firstly, the POF model was induced in mice by intragastric administration of Tripterygium wilfordii glycosides solution at 50 mg·kg~(-1) for 14 days. Ten days prior to the end of the modeling, the estrous cycle of mice was observed every day to evaluate the success of modeling. From the 1st day after modeling, the POF model mice were treated with QWGB by gavage every day and the treatment lasted four weeks. On the 2nd day after the end of the experiment, blood was collected from the eyeballs and the serum was separated by centrifugation. The ovaries and uterus were collected and the adipose tissues were carefully stripped. The organ indexes of the ovaries and uterus of each group were calculated. The serum estrogen(E_2) level of mice in each group was detected by ELISA. Protein samples were extracted from ovarian tissues of mice, and the differential proteins before and after QWGB intervention and before and after modeling were analyzed by quantitative proteomics using tandem mass tags(TMT). As revealed by the analysis of differential proteins, QWGB could regulate 26 differentially expressed proteins related to the POF model induced by T. wilfordii glycosides, including S100A4, STAR, adrenodoxin oxidoreductase, XAF1, and PBXIP1. GO enrichment results showed that the 26 differential proteins were mainly enriched in biological processes and cellular components. The results of KEGG enrichment showed that those differential proteins were involved in signaling pathways such as completion and coalescence cascades, focal adhesion, arginine biosynthesis, and terpenoid backbone biosynthesis. The complement and coalescence cascades signaling pathway was presumably the target pathway of QWGB in the treatment of POF. In this study, the proteomics technique was used to screen the differential proteins of QWGB in the treatment of POF in mice induced by T. wilfordii glycosides, and they were mainly involved in immune regulation, apoptosis regulation, complement and coagulation cascade reactions, cholesterol metabolism, and steroid hormone production, which may be the main mechanisms of QWGB in the treatment of POF.

TRAF4 Silencing Induces Cell Apoptosis and Improves Retinoic Acid Sensitivity in Human Neuroblastoma.

Neuroblastoma (NB) is a pediatric malignancy that arises in the peripheral nervous system, and the prognosis in the high-risk group remains dismal, despite the breakthroughs in multidisciplinary treatments. The oral treatment with 13-cis-retinoic acid (RA) after high-dose chemotherapy and stem cell transplant has been proven to reduce the incidence of tumor relapse in children with high-risk neuroblastoma. However, many patients still have tumors relapsed following retinoid therapy, highlighting the need for the identification of resistant factors and the development of more effective treatments. Herein, we sought to investigate the potential oncogenic roles of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family in neuroblastoma and explore the correlation between TRAFs and retinoic acid sensitivity. We discovered that all TRAFs were efficiently expressed in neuroblastoma, but TRAF4, in particular, was found to be strongly expressed. The high expression of TRAF4 was associated with a poor prognosis in human neuroblastoma. The inhibition of TRAF4, rather than other TRAFs, improved retinoic acid sensitivity in two human neuroblastoma cell lines, SH-SY5Y and SK-N-AS cells. Further in vitro studies indicated that TRAF4 suppression induced retinoic acid-induced cell apoptosis in neuroblastoma cells, probably by upregulating the expression of Caspase 9 and AP1 while downregulating Bcl-2, Survivin, and IRF-1. Notably, the improved anti-tumor effects from the combination of TRAF4 knockdown and retinoic acid were confirmed in vivo using the SK-N-AS human neuroblastoma xenograft model. In conclusion, the highly expressed TRAF4 might be implicated in developing resistance to retinoic acid treatment in neuroblastoma, and the combination therapy with retinoic acid and TRAF4 inhibition may offer significant therapeutic advantages in the treatment of relapsed neuroblastoma.

[Determination of flavonoids constituents in epimedium total flavone capsule and research on its anti-osteoporosis activity].

To develop a rapid resolution liquid chromatography (RRLC) method for the simultaneous determination of epimedoside A, epimedin A1, epimedin A, epimedin B, epimedin C, icariin, baohuosideⅡ, icarisideⅠ, sagittatoside B, 2"-O-rhamnosyl icarisideⅡ, and baohuosideⅠin epimedium total flavone capsule. At the same time, the effects of the above 11 compounds on osteogenic differentiation of MC3T3-E1 cells were investigated by detecting the content of alkaline phosphatase (AKP). The results showed that baohuoside Ⅱ had the highest activities, and both the activities of baohuoside Ⅱ and icariside Ⅰ were stronger than those of icariin.In this study, the content determination method of flavonoid glycosides was established, and the anti-osteoporosis effect of monomers was compared, providing technical support for the study of the pharmacodynamic and mechanism of Epimedium total flavone capsule.

[Study on difference of flavonoids content in stems and leaves of Mentha Haplocalycis Herba in different harvest periods].

The dynamic changes of active components in stems and leaves of Mentha Haplocalycis Herba(mint) at different harvest periods were investigated, and the optimum harvest time of mint was explored. In this study, hesperidin, diosmin, didymin and buddleoside were selected as flavonoids index components of mint, and the QAMS method was established to measure the contents of these flavonoids in mint. The contents of 4 flavonoid glycosides in the mint stems and leaves from three habitats harvested in different time were studied and evaluated comprehensively using statistical analysis and principal component analysis (PCA). The results showed that the contents of 4 components in the leaves are higher than that in the stems despite of habitats and harvest time, and they all exhibited dynamic changes along with the harvest periods within the same habitat. Three harvest periods in mid April, mid September and late October scored higher in comprehensive evaluation in Jiangsu region, the genuine producing area of Mentha Haplocalycis Herba. Combined with the yield and contents of active compounds, the optimum harvest time of mint in Jiangsu region was mid September and late October, which is basically consistent with the traditional harvesting periods.

Analysis of Non-Volatile Chemical Constituents of Menthae Haplocalycis Herba by Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry.

Menthae Haplocalycis herba, one kind of Chinese edible herbs, has been widely utilized for the clinical use in China for thousands of years. Over the last decades, studies on chemical constituents of Menthae Haplocalycis herba have been widely performed. However, less attention has been paid to non-volatile components which are also responsible for its medical efficacy than the volatile constituents. Therefore, a rapid and sensitive method was developed for the comprehensive identification of the non-volatile constituents in Menthae Haplocalycis herba using ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap). Separation was performed with Acquity UPLC® BEH C18 column (2.1 mm × 100 mm, 1.7 µm) with 0.2% formic acid aqueous solution and acetonitrile as the mobile phase under gradient conditions. Based on the accurate mass measurement (<5 ppm), MS/MS fragmentation patterns and different chromatographic behaviors, a total of 64 compounds were unambiguously or tentatively characterized, including 30 flavonoids, 20 phenolic acids, 12 terpenoids and two phenylpropanoids. Finally, target isolation of three compounds named Acacetin, Rosmarinic acid and Clemastanin A (first isolated from Menthae Haplocalycis herba) were performed based on the obtained results, which further confirmed the deduction of fragmentation patterns and identified the compounds profile in Menthae Haplocalycis herba. Our research firstly systematically elucidated the non-volatile components of Menthae Haplocalycis herba, which laid the foundation for further pharmacological and metabolic studies. Meanwhile, our established method was useful and efficient to screen and identify targeted constituents from traditional Chinese medicine extracts.

The ecological filter system for treatment of decentralized wastewater.

A vertical flow constructed wetland was combined with a biological aerated filter to develop an ecological filter, and to obtain the optimal operating parameters: The hydraulic loading was 1.55 m3/(m2·d), carbon-nitrogen ratio was 10, and gas-water ratio was 6. The experimental results demonstrated considerable removal efficiency of chemical oxygen demand (COD), ammonia nitrogen (NH4+-N), total nitrogen (TN), and total phosphorus (TP) in wastewater by the ecological filter, with average removal rates of 83.79%, 93.10%, 52.90%, and 79.07%, respectively. Concentration of NH4+-N after treatment met the level-A discharge standard of GB18918-2002. Compared with non-plant filter, the ecological filter improved average removal efficiency of COD, NH4+-N, TN, and TP by 13.03%, 25.30%, 14.80%, and 2.32%, respectively: thus, plants significantly contribute to the removal of organic pollutants and nitrogen. Through microporous aeration and O2 secretion of plants, the ecological filter formed an aerobic-anaerobic-aerobic alternating environment; thus aerobic and anaerobic microbes were active and effectively removed organic pollutants. Meanwhile, nitrogen and phosphorus were directly assimilated by plants and as nutrients of microorganisms. Meanwhile, pollutants were removed through nitrification, denitrification, filtration, adsorption, and interception by the filler. High removal rates of pollutants on the ecological filter proved that it is an effective wastewater-treatment technology for decentralized wastewater of mountainous towns.

[Research on ultrafine grinding technology of improving dissolution rates of effective components in Sanjie Zhentong capsule].

The effects of ultrafine grinding on the dissolution rates of the effective components in Sanjie Zhentong capsule (SZC) were studied in this experiment. Fine and ultrafine powder of SZC intermediates were made by ordinary grinding and ultrafine grinding technology, and then granulated by wet granulation. SZC were prepared by fine powder, ultrafine powder and ultrafine granules, respectively. With resveratrol and loureirin B as investigated indexes, dissolution rates of the four intermediates in SZC were determined by cup method and HPLC. The dissolution rates of resveratrol in SZC prepared by fine powder, ultrafine powder and ultrafine granules were 26.11%, 63.27%, 67.49%, respectively; and the dissolution rates of loureirin B were 7.160%, 20.29%, 23.05%, respectively. The dissolution rate of resveratrol and loureirin B in SZC prepared by ultrafine granules was the best. D90 size of ultrafine grinding was 13.221 µm and could improve the dissolution rates of resveratrol and loureirin B in SZC.

Research on ultrafine grinding technology of improving dissolution rates of effective components in Sanjie Zhentong capsule / 中国中药杂志

The effects of ultrafine grinding on the dissolution rates of the effective components in Sanjie Zhentong capsule (SZC) were studied in this experiment. Fine and ultrafine powder of SZC intermediates were made by ordinary grinding and ultrafine grinding technology, and then granulated by wet granulation. SZC were prepared by fine powder, ultrafine powder and ultrafine granules, respectively. With resveratrol and loureirin B as investigated indexes, dissolution rates of the four intermediates in SZC were determined by cup method and HPLC. The dissolution rates of resveratrol in SZC prepared by fine powder, ultrafine powder and ultrafine granules were 26.11%, 63.27%, 67.49%, respectively; and the dissolution rates of loureirin B were 7.160%, 20.29%, 23.05%, respectively. The dissolution rate of resveratrol and loureirin B in SZC prepared by ultrafine granules was the best. D90 size of ultrafine grinding was 13.221 μm and could improve the dissolution rates of resveratrol and loureirin B in SZC.

[Optimization of one-step pelletization technology of Jiuwei Xifeng granules by response surface methodology].

Using the qualified rates of particles as the evaluation indexes, the impact tactors of one-step pelletization technology of Jiuwei Xifeng granules were selected from six factors by the Plackett-Burman experimental design and the levels of non-significant factors were identified. According to the Plackett-Burman experimental design, choosing the qualified rates of particles and angle of repose as the evaluation indexes, three levels of the three factors were selected by Box-Behnken of central composite design to optimize the experimental. The best conditions were as follows: the fluid extract was sprayed with frequency of 29 r . min-1, inlet air temperature was 90 °C, the frequency of fan was 34 Hz. Under the response surface methodology optimized scheme, the average experimental results are similar to the predicted values, and surface methodology could be used in the optimization of one-step pelletization for Chinese materia medica.

Optimization of one-step pelletization technology of Jiuwei Xifeng granules by response surface methodology / 中国中药杂志

Using the qualified rates of particles as the evaluation indexes, the impact tactors of one-step pelletization technology of Jiuwei Xifeng granules were selected from six factors by the Plackett-Burman experimental design and the levels of non-significant factors were identified. According to the Plackett-Burman experimental design, choosing the qualified rates of particles and angle of repose as the evaluation indexes, three levels of the three factors were selected by Box-Behnken of central composite design to optimize the experimental. The best conditions were as follows: the fluid extract was sprayed with frequency of 29 r . min-1, inlet air temperature was 90 °C, the frequency of fan was 34 Hz. Under the response surface methodology optimized scheme, the average experimental results are similar to the predicted values, and surface methodology could be used in the optimization of one-step pelletization for Chinese materia medica.

[Modulation on the P-glycoprotein in the jejunum by combined use of Glycyrrhiza inflata and Kansui].

To investigate the modulation on the P-glycoprotein in the jejunum by combined use of Glycyrrhiza inflata and Kansui with ussing chamber and rt-pcr, Rhodamine 123 (R123), a P-gp substrate and fluorescein sodium (CF), a model drug of non-P-gp substrate transported by a passive diffusion were taken as investigational drugs. Because these two drugs can be easily assayed and widely used in various research fields. The permeability of R123 or CF via Wistar rat jejunum membranes was evaluated by in vitro ussing chamber after oral administration of four different decoctions of Glycyrrhiza inflata and Kansui for 1 week. And the concentration of R123 or CF was determined by the fluorospectrophotometry in the receiving solution. Meanwhile the expression of mdr1a in P-glycoprotein was detected by real-time fluorescent quantitative PCR. After oral administration of combined decoction of the single drug, the absorptive directed permeability of R123 increased significantly (P < 0.01). On the other hand, Kansui and combine decoction of the two drugs also decrease the permeability of secretory directed transport (P < 0.05). No action of Glycyrrhiza inflata was found on the secretory transport of R123 [Papp = (2.56 +/- 0.38) x 10(-5), cm x s(-1)] across the jejunum tissues, while Papp of control group was found [Papp = (2.35 +/- 0.27) x 10(-5), cm x s(-1)]. After oral administration of Kansui decoction for 1 week and 2 weeks, the levels of mdr1a expression in Wistar rats were lower than that of the control group, but there were no significant difference in the results. Meanwhile, Glycyrrhiza inflata had no effect on transport of CF across the jejunum tissues, though the other three groups could decrease the permeability of CF, as compared with control group. Kansui may slightly inhibit P-glycoprotein function in the intestinal membrane. For another, some compositions in Kansui inhibit P-glycoprotein function, and some others strengthen the tight junction between cells in the intestinal membrane to decrease permeability of CF. As the inhibitory action to P-glycoprotein was enhanced by combination of Glycyrrhiza inflata and Kansui, based on the results, it may be one of the mechanisms of creating toxicity once co-administration of Glycyrrhiza inflata and Kansui.

Modulation on the P-glycoprotein in the jejunum by combined use of Glycyrrhiza inflata and Kansui / 药学学报

To investigate the modulation on the P-glycoprotein in the jejunum by combined use of Glycyrrhiza inflata and Kansui with ussing chamber and rt-pcr, Rhodamine 123 (R123), a P-gp substrate and fluorescein sodium (CF), a model drug of non-P-gp substrate transported by a passive diffusion were taken as investigational drugs. Because these two drugs can be easily assayed and widely used in various research fields. The permeability of R123 or CF via Wistar rat jejunum membranes was evaluated by in vitro ussing chamber after oral administration of four different decoctions of Glycyrrhiza inflata and Kansui for 1 week. And the concentration of R123 or CF was determined by the fluorospectrophotometry in the receiving solution. Meanwhile the expression of mdr1a in P-glycoprotein was detected by real-time fluorescent quantitative PCR. After oral administration of combined decoction of the single drug, the absorptive directed permeability of R123 increased significantly (P < 0.01). On the other hand, Kansui and combine decoction of the two drugs also decrease the permeability of secretory directed transport (P < 0.05). No action of Glycyrrhiza inflata was found on the secretory transport of R123 [Papp = (2.56 +/- 0.38) x 10(-5), cm x s(-1)] across the jejunum tissues, while Papp of control group was found [Papp = (2.35 +/- 0.27) x 10(-5), cm x s(-1)]. After oral administration of Kansui decoction for 1 week and 2 weeks, the levels of mdr1a expression in Wistar rats were lower than that of the control group, but there were no significant difference in the results. Meanwhile, Glycyrrhiza inflata had no effect on transport of CF across the jejunum tissues, though the other three groups could decrease the permeability of CF, as compared with control group. Kansui may slightly inhibit P-glycoprotein function in the intestinal membrane. For another, some compositions in Kansui inhibit P-glycoprotein function, and some others strengthen the tight junction between cells in the intestinal membrane to decrease permeability of CF. As the inhibitory action to P-glycoprotein was enhanced by combination of Glycyrrhiza inflata and Kansui, based on the results, it may be one of the mechanisms of creating toxicity once co-administration of Glycyrrhiza inflata and Kansui.

[Effect of Glycyrrhiza inflata and Daphne genkwa on permeabilities of rhodamine 123, a P-glycoprotein substrate across rat jejunum membranes in vitro].

OBJECTIVE: To investigate the modulation of Glycyrrhiza inflata and Daphne genkwa on the permeability characteristics of rhodamine 123 (R123), one P-glycoprotein (P-gp) substrate, across the jejunum membranes. And then approach the possible permeability mechanism of the drugs after co-administration of G. inflata and D. genkwa in gastrointestinal tract. METHOD: The permeability of R123 or fluorescein sodium (CF) via Wistar rat jejunum membranes was evaluated by in vitro diffusion chamber system after oral administration of four different decoctions and 0.9% sodium chloride (20 mL x kg(-1)) for 1 week. And the concentration of R123 or CF was determined by the fluorospectrophotometry. The apparent permeability coefficient (P(app)) was calculated by the equation P(app) = dQ/d(t) x (1/A x C0), where P(app) was expressed in cm/s, dQ/dT was the slope of the linear portion of the permeation curves, A was the diffusion area, and C0 was the initial concentration of rebamipide in the donor side, and then compare their differences were compared with control group. RESULT: After oral administration of G. inflata decoction, D. genkwa decoction and decoction of the combination of the previous decoctions, the absorptive directed transport of R123 was significantly increased (P < 0.05, compared with control group). On the other hand, D. genkwa could also decrease the permeability of secretory directed transport (P(app) = 2.98 +/- 0.59), while no action of G. inflata was found on the secretory transport of R123 ( P(app) = 5.24 +/- 3.98) across the jejunum tissues, while P(app) of control group was 4.38 +/- 1.18. Meanwhile, G. inflata had no effect on transport of CF across the jejunum tissues, though the other three groups could decrease the permeability of CF, as compared with control group. CONCLUSION: G. inflata may slightly inhibit P-glycoprotein function in the intestinal membrane, while D. genkwa may be a relatively strong inhibitor of P-gp. For another, some compositions in D. genkwa inhibit P-gp function, and some others strengthen the tight junction between cells in the intestinal membrane to decrease permeability of CF. As the inhibitory action to P-gp was enhanced by combination of G. inflata and D. genkwa, based on the results, it may be one of the mechanisms of creating toxicity once co-administration of G. inflata and D. genkwa.

Effect of Glycyrrhiza inflata and Daphne genkwa on permeabilities of rhodamine 123, a P-glycoprotein substrate across rat jejunum membranes in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the modulation of Glycyrrhiza inflata and Daphne genkwa on the permeability characteristics of rhodamine 123 (R123), one P-glycoprotein (P-gp) substrate, across the jejunum membranes. And then approach the possible permeability mechanism of the drugs after co-administration of G. inflata and D. genkwa in gastrointestinal tract.</p><p><b>METHOD</b>The permeability of R123 or fluorescein sodium (CF) via Wistar rat jejunum membranes was evaluated by in vitro diffusion chamber system after oral administration of four different decoctions and 0.9% sodium chloride (20 mL x kg(-1)) for 1 week. And the concentration of R123 or CF was determined by the fluorospectrophotometry. The apparent permeability coefficient (P(app)) was calculated by the equation P(app) = dQ/d(t) x (1/A x C0), where P(app) was expressed in cm/s, dQ/dT was the slope of the linear portion of the permeation curves, A was the diffusion area, and C0 was the initial concentration of rebamipide in the donor side, and then compare their differences were compared with control group.</p><p><b>RESULT</b>After oral administration of G. inflata decoction, D. genkwa decoction and decoction of the combination of the previous decoctions, the absorptive directed transport of R123 was significantly increased (P < 0.05, compared with control group). On the other hand, D. genkwa could also decrease the permeability of secretory directed transport (P(app) = 2.98 +/- 0.59), while no action of G. inflata was found on the secretory transport of R123 ( P(app) = 5.24 +/- 3.98) across the jejunum tissues, while P(app) of control group was 4.38 +/- 1.18. Meanwhile, G. inflata had no effect on transport of CF across the jejunum tissues, though the other three groups could decrease the permeability of CF, as compared with control group.</p><p><b>CONCLUSION</b>G. inflata may slightly inhibit P-glycoprotein function in the intestinal membrane, while D. genkwa may be a relatively strong inhibitor of P-gp. For another, some compositions in D. genkwa inhibit P-gp function, and some others strengthen the tight junction between cells in the intestinal membrane to decrease permeability of CF. As the inhibitory action to P-gp was enhanced by combination of G. inflata and D. genkwa, based on the results, it may be one of the mechanisms of creating toxicity once co-administration of G. inflata and D. genkwa.</p>