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Aroma compounds in fruits and related products are known to occur in free and glycosidically bound forms. In this study, free and bound volatiles in juices and wines of two kiwifruit cultivars, Actinidia deliciosa ‘Hayward’ and A. chinensis ‘Hort16A’, were analyzed by solid-phase microextraction-gas chromatography-mass spectrometry. Results showed that 67 free and 79 bound volatiles were found in juices and wines. Alcohols were the most abundant free volatiles, while the most representative bound volatiles were terpenoids. β-Damascenone and 4-methyl-3-penten-2-one released from aroma precursors were found in ‘Hort16A’ juice for the first time. Fruity, floral, and sweet flavors appeared to be the characteristic aroma in kiwifruit juices and wines. Principal component analysis showed that kiwifruit wines had a great aroma potential. Cluster analysis revealed that juices and wines had different profile of free volatiles, while similar bound volatile compositions were found. Moreover, abundant bound volatiles were discovered in these two kiwifruit pomace for the first time, indicating their potential applications in food and cosmetics industry.

Sepsis and septic shock are the main cause of mortality in intensive care units. The prevention and treatment of sepsis remains a significant challenge worldwide. The endothelial cell barrier plays a critical role in the development of sepsis. Aminophylline, a non-selective phosphodiesterase inhibitor, has been demonstrated to reduce endothelial cell permeability. However, little is known regarding the role of aminophylline in regulating vascular permeability during sepsis, as well as the potential underlying mechanisms. In the present study, the Slit2/Robo4 signaling pathway, the downstream protein, vascular endothelial (VE)-cadherin and endothelial cell permeability were investigated in a lipopolysaccharide (LPS)-induced inflammation model. It was indicated that, in human umbilical vein endothelial cells (HUVECs), LPS downregulated Slit2, Robo4 and VE-cadherin protein expression levels and, as expected, increased endothelial cell permeability in vitro during inflammation. After administration of aminophylline, the protein expression levels of Slit2, Robo4 and VE-cadherin were upregulated and endothelial cell permeability was significantly improved. These results suggested that the permeability of endothelial cells could be mediated by VE-cadherin via the Slit2/Robo4 signaling pathway. Aminophylline reduced endothelial permeability in a LPS-induced inflammation model. Therefore, aminophylline may represent a promising candidate for modulating vascular permeability induced by inflammation or sepsis.

Background The emergence of new molecular targeted drugs provides new prospects for the treatment of advanced breast cancer; the future therapeutic trend includes chemotherapy combined with molecular targeted therapy. Apatinib mesylate, a novel, small anti-angiogenic agent, highly selectively inhibits the activity of vascular endothelial growth factor receptor-2 tyrosine kinase. Apatinib mesylate also blocks the signaling of vascular endothelial growth factor binding to its receptor, thereby strongly inhibiting tumor angiogenesis and exerting an anti-tumor effect. However, there have been no reports of a randomized controlled clinical trial of apatinib combined with vinorelbine for the treatment of triple-negative breast cancer (TNBC). We will compare the therapeutic effect of vinorelbine alone or in combination with apatinib mesylate, in patients with recurrent or metastatic TNBC in North China who have received at least two drug treatments, including anthracyclines and taxanes. Methods/analysis This study is a triple-blind, randomized, placebo-controlled, parallel-group clinical trial. We plan to include 238 female patients with locally recurrent or metastatic TNBC, admitted to the Liaoning Cancer Hospital & Institute, Northeast China. All enrolled patients will be randomized to oral vinorelbine alone (40 mg, thrice a week (Mondays, Wednesdays, and Fridays) in each 3-week cycle), or in combination with oral apatinib mesylate (500 mg, once daily in each 3-week cycle). Radiographic assessment will be performed every 6 weeks for 36 weeks and every 9 weeks thereafter. The primary outcome is progression-free survival and secondary outcomes include overall survival, disease control rate, objective response rate, and incidence of adverse events at grades 3 and 4, as defined by the National Cancer Institute Common Toxicity Criteria Version 4.0. Outcome measures will be evaluated at baseline ( Discussion Based on the data from this trial, we hope to identify a treatment plan that is suitable for female patients with TNBC, who have been treated with anthracyclines and taxanes, in Northeast China. Trial registration ClinicalTrials.gov: NCT03932526. Registered on 30 April 2019.

Background: Breast cancer is one of the most prevalent diseases threatening women's health today. Indepth research on breast cancer (BC) pathogenesis and prevention and treatment methods are gradually receiving attention. Chidamide is a novel histone deacetylase inhibitor (HDACi) that depresses the function of histone deacetylase, consequently affecting the growth of BC cells through epigenetic modification. However, preclinical and clinical studies show that chidamide is ineffective in long-term treatment. We demonstrated in previous experiments that TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in BC cells and is significantly less non-toxic to normal cells than chidamide. Therefore, in this study, we treated BC cells with chidamide and TRAIL to explore a novel option to reduce the clinical toxicity through augmenting the sensitivity for BC cells. Methods and Results: Results from the MTT and cell viability assays indicated that the combination of chidamide and TRAIL in MCF-7 and MDA-MB-231 cells induced BC cell death, while maintaining a reduced concentration of chidamide. Autophagy assay and annexin V analysis showed that the autophagosome microtubuleassociated protein1light chain3-II (LC3-II) was abnormally increased and much more early and late phase of apoptotic cells appeared during chidamide and TRAIL induction. Anti-tumor assays in a BC tumor xenograft model displayed that the mixture of chidamide and TRAIL exhibited stronger effects on inhibiting tumor growth. The data from real-time PCR and western blotting showed that the cytotoxic effect correlated with the expressions of related apoptosis and autophagy factors. Conclusion: Our data are the first to demonstrate the synergistic effects of chidamide and TRAIL in BC cells, specifically, the pharmacological effects on cell death induction. These results lay a solid experimental and theoretical basis to solve the clinical resistance of chidamide.

Principal component analysis (PCA) and partial least squares (PLS) regression were applied to investigate the effect of glutathione-enriched inactive dry yeast (g-IDY) on the amino acids and volatile components of kiwi wine. Results indicated that the addition of g-IDY had positive effect on most amino acids of kiwi wine, especially glutamine and glycine. In case of pure juice fermentation, the concentrations of ethyl decanoate, 2-methylbutyric acid, trans-2-nonenal and hexyl butyrate had notably positive correlation with the addition of g-IDY. PLS regression indicated that the amino acids were highly interrelated to the volatile compositions, and glycine had the strongest positive impact on the concentrations of esters and total volatile components. This might explain the similar effect of g-IDY on the amino acids and volatile components of kiwi wine. Besides, PLS regression showed that E-nose was a good method to predict volatile compositions of kiwi wine, especially esters.

Fresh “Xuxiang” kiwifruit juice with or without pomace was used for fermentation with different levels of glutathione‐enriched inactive dry yeast (g‐IDY). After alcohol fermentation, the glutathione (GSH) content, color parameters, phenolic composition, and antioxidant activities of kiwi wine were determined. The results suggested that the addition of g‐IDY to kiwi juice increased GSH, glutathione disulfide, total GSH concentrations, and the total phenolic content of kiwi wine. No obvious effect on individual phenolic content was found in wine with g‐IDY. The color parameters of wines fermented with pomace were remarkably improved with addition of 100 mg/L and 300 mg/L g‐IDY. The supply of g‐IDY improved the antioxidant activities of kiwi wine fermented with pomace. Principal component analysis suggested that with the same supplies of g‐IDY, wine fermented with pomace was better than wine fermented through pure juice in all measurements. PRACTICAL APPLICATIONS: The improvements in color and antioxidant capacity of kiwi wine have always been the focus of kiwi wine brewing. Constant improvement in methods which contribute to high quality of kiwi wine provides crucial support for wine industry and even agriculture development. The aim of this study is to investigate the impacts of glutathione‐enriched inactive dry yeast (g‐IDY) on quality of kiwi wine. These results could provide feasible advices about g‐IDY application to kiwi wine for improving the sensory and nutritional properties.

To enhance dissolution and improve oral bioavailability of the poorly water-soluble drug (andrographolide, ADP), using a natural triterpenoid glucoside (glycyrrhizin, GLC) as stabilizer, the ADP nanocrystals (ADP-NC) were prepared in water by homogenization method and converted into ADP nanocrystals-agglomerated particles (nanocomposite microparticles, ADP-NP) using spray-drying. Different types of matrix formers from glucoside (GLC), polyol (mannitol), disaccharide (lactose), polysaccharide (maltodextrin), and polymer (PVPK30) were systematically investigated their effects on particles properties of ADP-NP. And the acceptable ADP-NP was further characterized for oral bioavailability. The results demonstrated that ADP–NC with particles size of 476 ± 22 nm and span of 2.134 ± 0.016 were successfully prepared. The ADP-NP containing 100% ratio of GLC possessed a higher yield (60.47%), more excellent redispersibility (1.06) and flowability (HR 1.16), compared to those containing mannitol, lactose, PVPK30 and maltodextrin as matrix formers. These were related with the glass transition property of mannitol, lactose, PVPK30 and maltodextrin. The DSC and XRD analysis demonstrated that the mannitol, lactose, PVPK30 and maltodextrin were amorphous state presented in ADP-NP, but the crystal state of ADP and GLC remained unchanged during the preparation. The ADP-NP containing GLC showed the fast dissolution rate and significantly improved the bioavailability (794.338 ± 229.259 μg/L·h) of ADP. This study demonstrated the feasibility of nanocomposite microparticles strategy to improve the oral bioavailability of poorly soluble ADP, and GLC can be an alternative matrix former of ADP-NP, which could prevent from irreversible agglomerates of ADP-NC during spray-drying.

To help baicalin pass across BBB and improve its targeting in brain, we designed a novel formulation strategy of baicalin nanocrystals that preferentially adsorbing apolipoprotein E (ApoE) and repelling protein adsorption of opsonins. Intravenous baicalin nanocrystals suspensions (BCL-NS) modified by different surfactant were prepared by high-pressure homogenization. The targeting potential of surface-modified BCL-NS with mean particles size of about 250 nm was assessed by in vitro protein adsorption studies using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), and further evaluated in vivo pharmacokinetics. The protein adsorption results showed that BCL-NS/TPGS, BCL-NS/TW80 and BCL-NS/TPGS + TW80 adsorbed very high amounts of apolipoproteins (ApoA-I, ApoA-Ⅱ, ApoA-IV, ApoC-III, ApoE, ApoJ) and relative low amounts of opsonins (fibrinogen, immunoglobulin heavy chain gamma, immunoglobulin light chain). The pharmacokinetics results demonstrated the AUC (0-∞) in brain of the BCL-NS/TW80 + TPGS was 6.67 times as high as that of the BCL solution, and 2.59 times as high as that of the BCL-NS/TW80. It could be attributed to the most ApoE and Apo J adsorption indicative of strong BBB penetration, and least IgG γ and fibrinogen loading minimizing the risk of hepatic uptake. Combination of TW80 and TPGS can be rational choice of surfactants of baicalin nanocrystals for brain-targeting mediated by ApoE adsorption.

The β-glucosidase derived from microorganisms has attracted worldwide interest for their industrial applications, but studies on β-glucosidases from Oenococcus oeni are rare. In this paper, catalytic mechanism of a novel β-glucosidase BGL0224 of Oenococcus oeni SD-2a was explored for the first time by kinetic parameters determination, fluorescence spectroscopy and quenching mechanism analysis, molecular dynamics simulation. The results indicated that BGL0224 had universal catalytic effect on different types of glycoside substrates, but the catalytic efficiencies were different. Fluorescence quenching analysis results suggested that the quenching processes between BGL0224 and seven kinds of substrates were predominated by the static quenching mechanism. A reasonable three-dimensional model of BGL0224 was obtained using the crystal structure of E.coli BglA as a template. The analysis results of molecular simulation (RMSD, Rg, RMSF and hydrogen bonding) showed that the composite system ‘BGL0224-pNPG’ was very stable after 40 ns. The catalytic process of BGL0224 acting on ‘p-Nitrophenyl β- d -glucopyranoside’ conformed to the double displacement mechanism. Two glutamic acid residues ‘Glu178 and Glu377’ played a vital role in the whole catalytic process. Overall, this study gave specific insights on the catalytic mechanism of BGL0224, which was of great significance for developing its potential applications in food industry.

β-glucosidase is widely used in food industry for its glycosidic hydrolysis properties. In this paper, performance of a novel β-glucosidase BGL0224 from Oenococcus oeni for aroma enhancement of Cabernet Sauvignon wines was explored for the first time. The main projects included electronic nose, gas chromatography-mass spectrometry and sensory analysis. Principal component analysis indicated that electronic nose had the ability to discriminate different treatments by responding specifically to volatile compounds. Adding BGL0224 before alcoholic fermentation significantly increased the “aromatic index” of Cabernet Sauvignon wines. Compared with the control group, adding BGL0224 increased more than 15 volatile aroma compounds of Cabernet Sauvignon wines. Furthermore, with the addition of BGL0224 before alcoholic fermentation, the concentrations of medium-chain fatty acid ethyl esters, long-chain fatty acid ethyl esters and terpenes in wines were 3004.19 ± 36.14 μg/L, 4090.37 ± 94.07 μg/L and 210.21 ± 8.98 μg/L respectively, which were all significantly higher than other treatments. Sensory analysis showed that BGL0224 increased “tropical fruity”, “sweet fruity” and “floral notes” characteristics while inhibited “citrus” characteristics of Cabernet Sauvignon wines. Overall, this study provided support for industrial application of BGL0224 by evaluating its performance for aroma enhancement of Cabernet Sauvignon wines.

The aim of this study was to evaluate probiotic properties of ten Streptococcus thermophilus strains (st1 to st10) isolated from pickles in China. These strains all had β-galactosidase activity, which laid foundation for studying their probiotic properties. In this study, the bile salt hydrolase activity, lysozyme resistance, tolerance to simulated gastric juice, bile salt tolerance, and bacterial adhesion capacity to the Caco-2 cells of these selected strains were detected in vitro conditions. The results indicated that the bile salt hydrolase activities of st2, st6, and st9 were higher than that for other strains. St10 showed the greatest lysozyme resistance (> 80% survival), followed by st9, st8, st7, st5, and st6. As for the tolerance to simulated gastric juice, st5 possessed the highest survival rate (35%), followed by st6 (30%). St6 was the best performer in both bile salt tolerance and bacterial adhesion capacity to the Caco-2 cells. The results of fluorescence microscope and electron microscope further confirmed previous studies and more intuitively demonstrated the st6 strain's tolerance to harsh environments. Overall, these strains were expected to possess beneficial properties and have the potentiality to be probiotics.

Persimmon tannin (PT) exhibits antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) isolated from retail pork. The involved molecular mechanisms were investigated for the first time using transcriptome and metabolome in this study. Results showed that subinhibitory concentration of PT (0.5 mg/ml) induced significant changes in MRSA at both transcriptional and metabolic levels, as 370 genes and 19 metabolites were differentially expressed. Bioinformatic analysis revealed that the varying genes and metabolites were mainly involved in pathways of membrane transport, amino acids, carbohydrate, and energy metabolism. The highlighted changes were those related to osmotic regulation, intracellular pH regulation, amino acid synthesis and metabolism, glycolysis, TCA cycle and iron metabolism, suggesting the multifaceted effects including cell membrane damage, amino acids limitation, energy metabolism disorder and iron deprivation induced by PT. The results provided insight into the anti-MRSA mechanism of PT, which is useful for PT’s development and application in food safety.

Dysregulated activation of the cyclin-dependent kinases (CDKs) 4/6, leading to uncontrolled cell division, is hallmark of cancers. Further study of the cell cycle will advance the cancer treatment. As powerful and effective drugs, inhibitors of CDK 4/6 have been widely used in clinical practice for several malignancies, particularly against breast cancers driven by the estrogen receptor (ER). Three CDK4/6 inhibitors, including palbociclib (PD0332991), ribociclib (LEE011) and abemaciclib (LY2835219), have been approved by the US Food and Drug Administration (FDA) for the treatment of hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced or metastatic breast cancer. However, CDK4/6 inhibitors act downstream of many mitogenic signaling pathways, and this has implications for resistance. It is worth to note that the mechanisms of resistance are not very clear. Up to now, a small number of preclinical and clinical studies have explored potential mechanisms of CDK4/6 inhibitors resistance in breast cancer. On this basis, rational and effective combination therapy is under development. Here we review the current knowledge about the mechanisms and efficacy of CDK4/6 inhibitors, and summarize data on resistance mechanisms to make future combination therapies more accurate and reasonable.

β-Glucosidases play an important role in food industry. Oenococcus oeni are typical lactic acid bacteria that initiate malolactic fermentation of wines. 35 β-glucosidases from O. oeni were selected and their conserved domains and evolutionary relationships were further explored in this study. The homology analysis results indicated that 35 β-glucosidases were basically derived from GH1 and GH3 family. A novel β-glucosidase was successfully expressed and characterized. The recombinant protein, referred to as BGL0224, consisted of a total 480 amino acids with an apparent molecular weight of 55.15 kDa and was classified as GH1 family. It achieved the highest activity at pH 5.0 and 50 °C. The activity and stability were significantly increased when 12% ethanol was supplemented to the enzyme. Using p-NPG as substrate, the Km, Vmax and Kcat of BGL0224 were 0.34 mM, 382.81 U/mg and 351.88 s−1, respectively. In all, BGL0224 has good application prospects in food industry.

Due to limited understanding about the effect of cryo/thermal strength from drying process on the redispersibility of drug nanocrystals, the impact of the different type and concentration of stabilizers and matrix formers on the redispersibility of nanocrystals were systematically investigated. Andro nanosuspensions were transformed into Andro nanocrystals (Andro-NC) via different drying process. The results demonstrated that the redispersibility of Andro-NC at the aggressive cryo-strength (meant higher freezing rate) was more excellent than those at conservative and moderate condition. Compared to the thermal stress from drying, the employed amount and type of stabilizers more dramatically affected the redispersibility of Andro-NCP during lyophilization. The HPMC-sucrose and HPMC-sorbitol system achieved excellent performance that protected Andro-NC from crystal growth during lyophilization. During spray-drying, the impacts of types and amounts of stabilizers on the redispersibility of Andro-NCP were more significant compared to those induced by the thermal stress conditions. The polymers HPMC, PVPK30 and MCCS with high Tg played an outstanding role in preventing the Andro-NCP from breakage during spray-drying, due to the firmly steric barrier effect of polymeric stabilizers. It is concluded that Andro-NCP is subjected to agglomeration or crystal growth due to cryo/thermal stresses during drying. The polymeric stabilizers are more effective to protect Andro-NCP from the cryo/thermal damage during solidification process, which behaved strong surface adsorption and high glass transition property at different solidification stress.

Optic atrophy 1 (OPA1)-related mitochondrial fusion and mitophagy are vital to sustain mitochondrial homeostasis under stress conditions. However, no study has confirmed whether OPA1-related mitochondrial fusion/mitophagy is activated by melatonin and, consequently, attenuates cardiomyocyte death and mitochondrial stress in the setting of cardiac ischemia-reperfusion (I/R) injury. Our results indicated that OPA1, mitochondrial fusion, and mitophagy were significantly repressed by I/R injury, accompanied by infarction area expansion, heart dysfunction, myocardial inflammation, and cardiomyocyte oxidative stress. However, melatonin treatment maintained myocardial function and cardiomyocyte viability, and these effects were highly dependent on OPA1-related mitochondrial fusion/mitophagy. At the molecular level, OPA1-related mitochondrial fusion/mitophagy, which was normalized by melatonin, substantially rectified the excessive mitochondrial fission, promoted mitochondria energy metabolism, sustained mitochondrial function, and blocked cardiomyocyte caspase-9-involved mitochondrial apoptosis. However, genetic approaches with a cardiac-specific knockout of OPA1 abolished the beneficial effects of melatonin on cardiomyocyte survival and mitochondrial homeostasis in vivo and in vitro. Furthermore, we demonstrated that melatonin affected OPA1 stabilization via the AMPK signaling pathway and that blockade of AMPK repressed OPA1 expression and compromised the cardioprotective action of melatonin. Overall, our results confirm that OPA1-related mitochondrial fusion/mitophagy is actually modulated by melatonin in the setting of cardiac I/R injury. Moreover, manipulation of the AMPK-OPA1-mitochondrial fusion/mitophagy axis via melatonin may be a novel therapeutic approach to reduce cardiac I/R injury.

To investigate the effect of reduced glutathione (GSH) on the quality characteristics of apple wine, 10, 20 and 30 mg/L of GSH were added to apple juice before alcoholic fermentation. Meanwhile, apple wine fermented by Saccharomyces cerevisiae which had been pre-incubated with GSH (100 mg/L) was another experimental group. Mono-phenols, GSH and oxidized glutathione (GSSG) were determined by HPLC. Aroma compounds were analysed by GC–MS. Further, E-nose was applied to monitor the odor. After fermentation, GSH content was the same in all of the samples. However, for the apple wine with GSH addition, GSSG content increased significantly. Notably, GSH could reduce the color index, protect chlorogenic acid and phloretin, decrease the content of epicatechin and catechin as well as change the profile of aroma compounds (higher levels of 2-methyl-1-propanol, 3-methyl-1-butanol, ethyl benzoate, linalool, etc.). GSH may be used for flavor enhancement and quality improvement of apple wine.

Understanding the mechanistic basis for temozolomide (TMZ)-induced glioma resistance is an important obstacle in developing an effective form of chemotherapy for this type of tumor. Glycogenolysis is known to play an essential role in cellular proliferation and potassium homeostasis and involves the glycogen phosphorylase isoenzyme BB (GPBB). In this investigation, plasma GPBB was correlated with TMZ-resistance. Elevated plasma GPBB concentrations were found to be more frequent in a TMZ-resistant cohort of patients with poor survival rates. TMZ inhibits cell proliferation and induces TMZ resistance by upregulating the expression of O(6)-methylguanine-DNA methyltransferase (MGMT). This process requires glycogenolysis, which was confirmed herein by treatment with 1,4-dideoxy-1,4-imino-D-arabinitol hydrochloride, a glycogenolysis inhibitor and a special GPBB inhibitor. Acute TMZ treatment leads to upregulation of [Ca2+]i, extracellular-regulated kinase (ERK)1/2 phosphorylation, and chronic TMZ treatment leads to upregulation of the expression of Na,K-ATPase, ERK1/2, and MGMT protein. Upregulation was abolished for each of these by inhibitors of transient receptor potential channel 1 and the inositol trisphosphate receptor. L-channel [Ca2+]i inhibitors and RyR antagonists had no such effect. These results demonstrate that [Ca2+]i-dependent glycogenolysis participates in acquired glioma TMZ-resistance by upregulating MGMT via a Na,K-ATPase/ERK1/2 signaling pathway. GPBB and glycogenolysis may therefore represent novel therapeutic targets for overcoming TMZ-resistant gliomas.

Yueqin Ma,1,2,* Yang Yang,2,* Jin Xie,1 Junnan Xu,1 Pengfei Yue,1 Ming Yang1 1Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi, 330004, 2Department of Medicine and Pharmacy, 94th Hospital of People’s Liberation Army, Nanchang, Jiangxi, 330002, China *These authors contributed equally to this work Objective: The current study sought to design a quickly dissolving, high drug loading nanocrystal-based solid dispersion (NC-SD) in order to improve the dissolution of poorly soluble drugs. Methods: The NC-SD was prepared by means of combination of homogenization and spray-drying. Polymer hydroxypropylmethylcellulose (HPMC) was used as baseline dispersant for NC-SD of the model drug – andrographolide (AG). Three superdisintegrants cohomogenized with HPMC were used as codispersant for AG-NC-SD and compared to common water-soluble dispersants – mannitol and lactose. The dissolution characterization and oral bioavailability of AG-NC-SD were evaluated. Results: The AG-NC-SD with the higher concentration of HPMC exhibited fast dissolution due to the enhanced wettability of HPMC. The water-soluble codispersants (mannitol and lactose) did not completely prevent AG-NC from aggregation during spray-drying. To achieve much faster AG release, cohomogenized superdisintegrants at a level of 20% must be used along with 25% HPMC. Compared with water-soluble dispersants like mannitol and lactose, superdisintegrants with high swelling capacity were much more effective dispersants for enhancing fast redispersion/dissolution of AG-NC-SD via a swelling-triggered erosion/disintegration mechanism. Surfactant-free AG-NC-SD with 15% cohomogenized sodium carboxymethyl starch combined with 15% HPMC and 10% lactose enhanced the dissolution further, without comprising drug loading, exhibited a barely compromised dissolution rate compared to precursor NC suspensions (f2>50), and possessed drug loading up to 67.83%±1.26%. The pharmacokinetics results also demonstrated that the AG-NC-SD significantly improved the bioavailability in vivo of AG (P

Radix Sophorae flavescentis is generally used for the treatment of different stages of prostate cancer in China. It has ideal effects when combined with surgical treatment and chemotherapy. However, its active components are still ambiguous. We devised a comprehensive two-dimensional PC-3 prostate cancer cell membrane chromatography system for screening anti-prostate cancer components in Radix Sophorae flavescentis. Gefitinib and dexamethasone were chosen as positive and negative drugs respectively for validation and optimization the selectivity and suitability of the comprehensive two-dimensional chromatographic system. Five compounds, sophocarpine, matrine, oxymatrine, oxysophocarpine, and xanthohumol were found to have significant retention behaviors on the PC-3 cell membrane chromatography and were unambiguously identified by time-of-flight mass spectrometry. Cell proliferation and apoptosis assays confirmed that all five compounds had anti-prostate cancer effects. Matrine and xanthohumol had good inhibitory effects, with half maximal inhibitory concentration values of 0.893 and 0.137 mg/mL, respectively. Our comprehensive two-dimensional PC-3 prostate cancer cell membrane chromatographic system promotes the efficient recognition and rapid analysis of drug candidates, and it will be practical for the discovery of prostate cancer drugs from complex traditional Chinese medicines.

The cotransporter of Na(+) , K(+) , 2Cl(-) , and water, NKKC1, is activated under two conditions in the brain, exposure to highly elevated extracellular K(+) concentrations, causing astrocytic swelling, and regulatory volume increase in cells shrunk in response to exposure to hypertonic medium. NKCC1-mediated transport occurs as secondary active transport driven by Na(+) /K(+) -ATPase activity, which establishes a favorable ratio for NKCC1 operation between extracellular and intracellular products of the concentrations of Na(+) , K(+) , and Cl(-) × Cl(-) . In the adult brain, astrocytes are the main target for NKCC1 stimulation, and their Na(+) /K(+) -ATPase activity is stimulated by elevated K(+) or the β-adrenergic agonist isoproterenol. Extracellular K(+) concentration is normal during regulatory volume increase, so this study investigated whether the volume increase occurred faster in the presence of isoproterenol. Measurement of cell volume via live cell microscopic imaging fluorescence to record fluorescence intensity of calcein showed that this was the case at isoproterenol concentrations of ≥1 µM in well-differentiated mouse astrocyte cultures incubated in isotonic medium with 100 mM sucrose added. This stimulation was abolished by the β1 -adrenergic antagonist betaxolol, but not by ICI118551, a β2 -adrenergic antagonist. A large part of the β1 -adrenergic signaling pathway in astrocytes is known. Inhibitors of this pathway as well as the glycogenolysis inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol hydrochloride and the NKCC1 inhibitors bumetanide and furosemide abolished stimulation by isoproterenol, and it was weakened by the Na(+) /K(+) -ATPase inhibitor ouabain. These observations are of physiological relevance because extracellular hypertonicity occurs during intense neuronal activity. This might trigger a regulatory volume increase, associated with the post-excitatory undershoot.

The objective of this study is to prepare a novel drug nanosuspension modified by a natural triterpenoid saponin (glycyrrhizin (GZ)) and evaluate its stability and redispersibility. A poorly soluble drug (andrographolide (AGE)) was used as a model drug. AGE nanosuspensions (AGE-NS) using GZ as natural stabilizer with mean particle size of 487 nm were firstly prepared by homogenization and converted into dried AGE nanosuspension powder (AGE-NP) by freeze-drying. It was found that GZ was able to prevent the aggregation of AGE nanocrystals and the freeze-dried AGE-NP could easily redisperse back to AGE-NS. It was related with special properties of GZ that possessed the interfacial property (37.02 ± 0.29 N/m) and electrostatic effect (−43.6 ± 0.9 mV) and could entrap AGE nanocrystals into its network structure. The freeze-dried AGE-NP/GZ exhibited excellent performance, compared with those combined with trehalose as matrix formers. The powder X-ray diffraction result demonstrated that GZ did not alter the AGE crystal state. The dissolution of AGE-NP/GZ (99.87%) was significantly enhanced, compared with the coarse AGE (42.35%). This study demonstrated that GZ could be used as a novel multifunctional stabilizer for production of drug nanosuspensions and provided a promising basis for further formulation development of poorly soluble drug.

This study is aimed at seeking a nature saponin-based stabilizer for drug nanosuspensions. A poorly soluble drug (baicalein, BCL) was used as a model drug. BCL nanosuspensions with particle size of 156 nm were prepared by means of homogenization and converted into BCL nanocrystals (BCL-NC) stabilized with panax notoginseng saponins (PNS). It was found that PNS was able to prevent the aggregation of BCL-NS during storage and improve the redispersibility of BCL-NC after freeze-drying and spray-drying, compared with polymer stabilizer PVPK30. The freeze-dried and spray-dried BCL-NC with PNS exhibited excellent performance as evidenced by scanning_electron_microscope (SEM) analysis. It was the reason that PNS possessed the interfacial property (41.69 ± 0.32 mN/m) and electrostatic effect (−40.1 ± 1.6 mV), which could easily adsorb onto the surface of hydrophobic BCL nanocrystals and prevent from its aggregation. It is concluded that PNS can be used as an effective nature stabilizer for production of drug nanocrystals.

Poorly water-soluble drugs offer challenges in developing a formulation product with adequate bioavailability. This study took advantage of the features of nanocrystals and direct compression technologies to develop a novel solid self-nanodispersion delivery system for andrographolide (Andro) in order to increase its dissolution rate for enhancing bioavailability. Andro nanosuspensions (Andro-NS) with a particle size of about 500 nm were prepared by homogenization technology and further converted into dried nanocrystal particles (Andro-NP) via spray-drying. The solid self-nanodispersion delivery system (Andro-SNDS)-loaded Andro-NP was prepared via direct compression technology. The DSC and PXRD results demonstrated that the Andro nanocrystals retained its original crystallinity. The dissolution of the Andro-SNDS formulation was 85.87% in pure water over 30 min, better than those of the coarse Andro and physical mixture of Andro and stabilizer. And the C max (299.32 ± 78.54 ng/mL) and AUC0-∞ (4440.55 ± 764.13 mg/L · h) of the Andro-SNDS formulation were significantly higher (p

This study investigates the role of the intracellular adenosine transporter equilibrative nucleoside transporter 3 (ENT3) in stimulated release of the gliotransmitter adenosine triphosphate (ATP) from astrocytes. Within the past 20 years, our understanding of the importance of astrocytic handling of adenosine, its phosphorylation to ATP, and release of astrocytic ATP as an important transmitter has become greatly expanded. A recent demonstration that the mainly intracellular nucleoside transporter ENT3 shows much higher expression in freshly isolated astrocytes than in a corresponding neuronal preparation leads to the suggestion that it was important for the synthesis of gliotransmitter ATP from adenosine. This would be consistent with a previously noted delay in transmitter release of ATP in astrocytes but not in neurons. The present study has confirmed and quantitated stimulated ATP release in response to glutamate, adenosine, or an elevated K+ concentration from well-differentiated astrocyte cultures, measured by a luciferin–luciferase reaction. It showed that the stimulated ATP release was abolished by downregulation of ENT3 with small interfering RNA (siRNA), regardless of the stimulus. The concept that transmitter ATP in mature astrocytes is synthesized directly from adenosine prior to release is supported by the postnatal development of the expression of the vesicular transporter SLC17A9 in astrocytes. In neurons, this transporter carries ATP into synaptic vesicles, but in astrocytes, its expression is pronounced only in immature cells and shows a rapid decline during the first 3 postnatal weeks so that it has almost disappeared at the end of the third week in well-differentiated astrocytes, where its role has probably been taken over by ENT3.

Both ATP and glutamate are on one hand essential metabolites in brain and on the other serve a signaling function as transmitters. However, there is the major difference that the flux in the pathway producing transmitter glutamate is comparable to the rate of glucose metabolism in brain, whereas that producing transmitter ATP is orders of magnitude smaller than the metabolic turnover between ATP and ADP. Moreover, de novo glutamate production occurs exclusively in astrocytes, whereas transmitter ATP is produced both in neurons and astrocytes. This chapter deals only with ATP and exclusively with its formation and release in astrocytes, and it focuses on potential associations with glycogenolysis, which is known to be indispensable for the synthesis of glutamate. Glycogenolysis is dependent upon an increase in free intracellular Ca2+ concentration (Ca2+]i). It can be further stimulated by cAMP, but in contrast to widespread beliefs, cAMP can on its own not induce glycogenolysis. Astrocytes generate ATP from accumulated adenosine, and this process does not seem to require glycogenolysis. A minor amount of the generated ATP is utilized as a transmitter, and its synthesis requires the presence of the mainly intracellular nucleoside transporter ENT3. Many transmitters as well as extracellular K+ concentrations high enough to open the voltage-sensitive L-channels for Ca2+ cause a release of transmitter ATP from astrocytes. Adenosine and ATP induce release of ATP by action at several different purinergic receptors. The release evoked by transmitters or elevated K+ concentrations is abolished by DAB, an inhibitor of glycogenolysis.

Infarct size and brain edema following ischemia/reperfusion are reduced by inhibitors of the Na+, K+, 2Cl−, and water cotransporter NKCC1 and byβ1-adrenoceptor antagonists. NKCC1 is a secondary active transporter, mainly localized in astrocytes, driven by transmembrane Na+/K+gradients generated by the Na+,K+-ATPase. The astrocytic Na+,K+-ATPase is stimulated by small increases in extracellular K+concentration and by theβ-adrenergic agonist isoproterenol. Larger K+increases, as occurring during ischemia, also stimulate NKCC1, creating cell swelling. This study showed no edema after 3 hr medial cerebral artery occlusion but pronounced edema after 8 hr reperfusion. The edema was abolished by inhibitors of specificallyβ1-adrenergic pathways, indicating failure of K+-mediated, but notβ1-adrenoceptor-mediated, stimulation of Na+,K+-ATPase/NKCC1 transport during reoxygenation. Ninety percent reduction of extracellular Ca2+concentration occurs in ischemia. Ca2+omission abolished K+uptake in normoxic cultures of astrocytes after addition of 5 mM KCl. A large decrease in ouabain potency on K+uptake in cultured astrocytes was also demonstrated in Ca2+-depleted media, and endogenous ouabains are needed for astrocytic K+uptake. Thus, among the ionic changes induced by ischemia, the decrease in extracellular Ca2+causes failure of the high-K+-stimulated Na+,K+-ATPase/NKCC1 ion/water uptake, makingβ1-adrenergic activation the only stimulus and its inhibition effective against edema.

We agree with [Jin et al. (2013)][1] that astrocytes accumulate extracellular K+. They discuss how differences in (a) resting extracellular space (ECS) volume, (b) diffusion-limited water/K+ transport, and (c) ECS contraction during K+ reuptake may differently affect astrocytic K+ uptake in wild

Neuronal excitation leads to an increase of the extracellular K(+) concentration ([K(+)]o) in brain. This increase has at least two energy-consuming consequences: (1) a depolarization-mediated change in intracellular pH (pHi) in astrocytes due to depolarization-mediated increased activity of the acid-extruding Na(+)/bicarbonate transporter NBCe1 (driven by secondary active transport, supported by ion gradients established by the Na(+), K(+)-ATPase); and (2) activation of cellular reuptake of K(+) mediated by the Na(+), K(+)-ATPase in both neurons and astrocytes. Astrocytic, but not neuronal increase in NBCe1 activity and pHi is also seen after chronic treatment with either of the two anti-bipolar drugs carbamazepine or valproic acid. The third 'classical' anti-bipolar drug, 'lithium' increases astrocytic pHi by a different mechanism (stimulation of the acid extruding Na(+)/H(+) exchanger NHE1). The acid extruder fluxes, which depend upon the change in pHi per time unit (ΔpHi/Δt) and intracellular buffering power, have not been established in most of these situations. Therefore their stimulatory effects on energy metabolism has not been quantitated. This has been done in the present study in cultured mouse astrocytes. pHi was determined using the fluorescent pH-sensitive indicator BCECF-AM and an Olympus IX71 live cell imaging fluorescence microscope. Molar acid extrusion fluxes (indicating transporter activity) were determined as pHi changes/min during recovery after acid-loading with NH3/NH4 (+), NBCe1 mRNA and protein expression in the cultured cells by, respectively RT-PCR and Western blotting. Drug-induced up-regulation of acid extrusion flux was slow and less than physiologically seen after increase in K(+) concentration. Energetically, K(+) uptake is much costlier than NBCe1 activity.

The involvement of glycogenolysis, occurring in astrocytes but not in neurons, in learning is undisputed (Duran et al., JCBFM, in press). According to one school of thought the role of astrocytes for learning is restricted to supply of substrate for neuronal oxidative metabolism. The present ‘perspective’ suggests a more comprehensive and complex role, made possible by lack of glycogen degradation, unless specifically induced by either i) activation of astrocytic receptors, perhaps especially beta-adrenergic, or ii) even small increases in extracellular K+ concentration above its normal resting level. It discusses i) the known importance of glycogenolysis for glutamate formation, requiring pyruvate carboxylation; ii) the established role of K+-stimulated glycogenolysis for K+ uptake in cultured astrocytes, which probably indicates that astrocytes are an integral part of cellular K+ homeostasis in the brain in vivo; and iii) the plausible role of transmitter-induced glycogenolysis, stimulating Na+,K+-ATPase/NKCC1 activity and thereby contributing both to the post-excitatory undershoot in extracellular K+ concentration and the memory-enhancing effect of transmitter-mediated reduction of slow neuronal afterhyperpolarization (sAHP).