Rapid Commun Mass Spectrom. 2009 Jul 28; 23(17): 2724-2732Wang Y, Ding C, Du K, Xiao Y, Wu C, Zhang J, Qin H, Du GXiao-xu-ming decoction (XXMD) prescription is a traditional Chinese prescription that has been widely used to treat theoplegia and the sequela of theoplegia. Modern pharmacological research has also indicated that the active fraction from XXMD is able to treat cardiovascular diseases and Alzheimer's disease. In the study reported here, high-performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry (HPLC/FTICR-MS) was developed to identify active compounds and their metabolites after oral administration of active fraction from Xiao-xu-ming decoction to rats, using parent mass list triggered data-dependent multiple-stage mass analysis at a resolving power of 100 000 in the external calibration mode. The mass accuracies obtained for full-scan MS were within 2 ppm in most cases. Fifteen constituents were identified in the active fraction from XXMD and the biological samples of rats. The fragmentation behaviors of these constituents were summarized which would be helpful for structural characterization. The profiles of the constituents in the active fraction and biological samples of rats were obtained which provided us with much information for a better understanding of the chemical basis of the pharmacologic actions of XXMD. Copyright (c) 2009 John Wiley & Sons, Ltd.

Angew Chem Int Ed Engl. 2009 Jul 23; Zhao L, Xie F, Cheng G, Hu Y

Biomol NMR Assign. 2009 Jun; 3(1): 95-7Zhang F, Guo C, Lou L, Lin DLipocalin2 plays an important role in the innate immune system. In this article we report the backbone and side-chain resonance assignments of rat lipocalin2 (rLcn2). These assignments provide a basis for determining the structure and dynamics of rLcn2.

Zhongguo Zhong Yao Za Zhi. 2009 Apr; 34(7): 861-3Liu A, Xu L, Zou Z, Yang SOBJECTIVE: To isolate and identify chemical constituents of Cassia alata. METHOD: Compounds were separated and purified by column chromatography with silica gel and Sephadex LH-20, elucidated by spectroscopic methods including 1D and 2D NMR, IR and MS techniques. RESULT: Twelve compounds were isolated from C. alata, which were identified as chrysoeriol (1), kaempferol (2), quercetin (3), 5,7,4'-trihydroflavanone (4), kaempferol-3-O-beta-D-glucopyranoside (5), kaempferol-3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (6), 17-hydrotetratriacontane (7), n-dotriacontanol (8), n-triacontanol (9), palmitic acid ceryl ester (10), stearic acid (11), palmitic acid (12). CONCLUSION: Compounds 3-12 were isolated from C. alata for the first time.

Nat Prod Commun. 2009 Jun; 4(6): 825-30Huang HL, Zhang JQ, Chena GT, Lu ZQ, Sha N, Guo DAOxyresveratrol (trans-2,4,3',5'-tetrahydroxystilbene, OXY) and resveratrol (trans-3,5,4'-trihydroxystilbene, RES) are the two most important constituents of the traditional Chinese medicine Smilax china. A reversed-phase high-performance liquid chromatographic method was developed to determine OXY and RES in rat bile and urine after oral administration of Smilax china extract. The biological samples were analyzed by HPLC on Aglient Zorbax SB-C18 column (250 x 4.6 mm, 5 microm) at a wavelength 320 nm and at a flow rate of 1.0 mL/min. The method was accurate and reproducible for determination. The cumulative excretion of OXY and RES was 0.29% and 0.97% in bile samples, 0.84% and 0.65% in urine samples, respectively.

Nat Prod Commun. 2009 Jun; 4(6): 813-8Sha N, Huang HL, Zhang JQ, Chen GT, Tao SJ, Yang M, Li XN, Li P, Guo DAA simple, sensitive and specific high-performance liquid chromatography-UV (HPLC-UV) method has been developed to simultaneously quantify the eight major bioactive phenolic compounds in Chinese propolis, namely caffeic acid, isoferulic acid, 3,4-dimethoxycinnamic acid, pinobanksin 5-methyl ether, pinocembrin, benzyl caffeate, chrysin and galangin. This HPLC assay was performed on an Agilent Zorbax Extend-C18 (250 x 4.6 mm, 5 microm) column with a gradient of methanol and 0.2% aqueous acetic acid (v/v) in 50 min, at a flow rate of 1.0 mL/min, and detected at 290 nm. All calibration curves showed good linearity (r2 > 0.999) within the test ranges. The intra- and inter-day assay precision (RSD) of eight phenolic compounds were in the range of 0.07-4.92%. The recoveries were between 98.3% and 104.8%. This assay was applied to the evaluation of nineteen samples from different origins in China. The results indicated that the developed assay could be readily utilized for the quality control of propolis.

Nat Prod Commun. 2009 Jun; 4(6): 777-82Chong W, Feng XY, Zhen GZ, Dan L, Yue DThe objective of the present study was to investigate the effects of saponins from the anomalous fruits of Gleditsia sinensis on mast cell degranulation triggered by compound 48/80 and to determine possible structure-activity relationships. Among the eleven saponins tested, four effectively inhibited beta-hexosaminidase release from rat peritoneal mast cells. Studies of structure-activity relationships indicated that saponins with an aglycone of echinocystic acid instead of oleanolic acid were more effective. A sugar chain at C-3 was essential for the inhibitory effects, and a single sugar chain was the most effective. The length and structures of the oligosaccharide chain at C-28 were also critical for the activity of the compounds, and introduction of monoterpene units to the oligosaccharide chain substantially decreased the activity. To gain insight into the mechanisms responsible for preventing mast cell degranulation, the effects of saponins on intracellular cAMP were examined. After preincubation with mast cells for different times, the four active saponins significantly increased the intracellular cAMP content. These findings suggested that the four saponins might be the active constituents of the anomalous fruits of G. sinensis for antiallergic activities, and they prevented mast cell degranulation probably by elevating intracellular cAMP levels.

Zhongguo Zhong Yao Za Zhi. 2009 Apr; 34(7): 854-7Li Y, Yang S, Bai SOBJECTIVE: To investigate the chemical constituents of the roots of Angelica polymorpha. METHOD: Silica gel column chromatography was employed for the isolation and purification of chemical constituents. The structures were identified on the basis of spectral data and chemical evidence. RESULT: Fourteen compounds were isolated and identified as follows, 5-hydroxy-2-[(angeloyloxy) methyl] furan [3', 2': 6, 7] chromone (1), octacosanoic acid (2), isoimperatorin (3), 3'S-(-)-O-acetylhamaudol (4), bergapten (5), iso-oxypeucedanin (6), beta-sitosterol (7), angeliticin A (8) , saxalin (9), pabulenol (10), noreugenin (11), oxypeucedanin hydrate (12), daucosterol (13), sucrose (14). CONCLUSION: Compound 1 is a new chromone, named polymorchromone A. Compounds 2, 4, 11, 13, 14 were isolated from A. polymorpha for the first time.

Zhongguo Zhong Yao Za Zhi. 2009 Apr; 34(7): 924-8Hua Y, Huang L, Chen M, Xiao PThe authors propose suggestions for definition of classification position, classification standard, nomenclature and naming methods of chemotype on discussing the significance of chemotype of medicinal plants. This classification of chemotype should be established in infraspecific categories of "forma". Chemotype identification mainly has two aspects. One is that the main constituents are distinct or one or two components are half or more than half of the total chemical content. The main constituents come from the same biosynthetic pathway and have some genetic stability. The other is the chemical variation is genetic. The chemotype of medicinal plants study on the classification has important theoretical and practical value for quality assessment, resource development and the genuine medicinal research. It also can ensure the safe and effective of clinical medicine.

Zhongguo Zhong Yao Za Zhi. 2009 Apr; 34(7): 807-11Lv J, Guo L, Huang L, Liang L, Sun Y, Zhang X, Han X, Zhang HFor optimal adaptive cultivation region selection, we used ecology factors characterized Duolun region as model area to carry out the adaptive habitat division of Paeonia lactiflora. Similar priority comparison of ecology factors.in 91 cities were calculated by Fuzzy methods, then, distance of the ecology factors were transferred to spacial model by geography information system (,GIS) and modified by soil utilization map of China. The results showed that P. lactiflora were mainly distributed in the Daxing'an Mountain, Changbaishan and qinling range which were divided into six grades of suitable regions belonging to three geographical distributed units. The most similar areas to Duolun were Huade, Xilinhaote, Suolun and Zhangbei. P. lactiflora's distribution and quality are relevant with longitude and latitude, and temperature and rainfall.

Zhongguo Zhong Yao Za Zhi. 2009 Mar; 34(6): 744-7Jia Y, Yi H, Pen B, Li J, Yang HOBJECTIVE: To explore the anti-inflammatory effect and possible mechanism of Shuang-huang-lian (SHL) microemulsion. METHOD: Rat model of pleuritis was established by thoracic injecting 0.2 mL of 1% carrageenan. Rats in the treated groups were orally administered with SHL microemulsion prescription 1, 2, and oral liquid, while those in the positive control group were given aspirin. Rats in the normal group and the model group were given equal volume of water. Each groups were given their medicine for successive 6 days. Modeling was performed 30 mins after the 5th day medication. After 12 hrs of modeling, took suction of the pleurorrhea and measured the amount of tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8), prostaglandin E2 (PGE2) and protein (pro). RESULT: Compared with the normal group, all the parameters were higher in model group (TNF-alpha and IL-8 P

Biomed Chromatogr. 2009 Jul 23; Geng F, He Y, Yang L, Wang ZAngiotensin-converting enzyme (ACE) plays an important role in the renin-angiotensin system and ACE activity is usually assayed in vitro by monitoring the transformation from a substrate to the product catalyzed by ACE. A rapid and sensitive analysis method or ACE activity by quantifying simultaneously the substrate hippuryl-histidyl-leucine and its product hippuric acid using an ultra-performance liquid chromatography coupled with electrospray ionization-mass spectrometry (UPLC-MS) was first developed and applied to assay the inhibitory activities against ACE of several natural phenolic compounds. The established UPLC-MS method showed obvious advantages over the conventional HPLC analysis in shortened running time (3.5 min), lower limit of detection (5 pg) and limit of quantification (18 pg), and high selectivity aided by MS detection in selected ion monitoring (SIM) mode. Among the six natural products screened, five compounds, caffeic acid, caffeoyl acetate, ferulic acid, chlorogenic acid and resveratrol indicated potent in vitro ACE inhibitory activity with IC(50) values of 2.527 +/- 0.032, 3.129 +/- 0.016, 10.898 +/- 0.430, 15.076 +/- 1.211 and 6.359 +/- 0.086 mm, respectively. A structure-activity relationship estimation suggested that the number and the situation of the hydroxyls on the benzene rings and the acrylic acid groups may play the most predominant role in their ACE inhibitory activity. Copyright (c) 2009 John Wiley & Sons, Ltd.

Zhongguo Zhong Yao Za Zhi. 2009 Apr; 34(7): 884-8Dong Y, Zhang Y, Yang Q, Li Y, Zhu XOBJECTIVE: To research the intestinal absorption characteristics of paeoniflorin in extractive Radix Paeoniae Alba in the different intestinal segment, and the interaction with P-glycoprotein. METHOD: Paeoniflorin, a representative component in extractive Radix Paeoniae Alba, on the intestinal absorption was studied in vitro using everted gut sacs model and detected by HPLC method. The absorption characteristics was evaluated by the absorption parameter. RESULT: The absorption of paeoniflorin was linearity at different intestine segment and dose, and the square of regrees correlation coefficient exceed 0.9 (R2 > 0.9), which consistent with zero order rate process. The Kalpha of paeoniflorin showed a dose-dependent increase along with the raised dose of extractive Radix Paeoniae Alba, indicated it was a mechanism of passive absorption. The absorption rate was jejunum > ileum > colon. Verapamil (100 micromol x L(-1)), a inhibitor of the P-glycoprotein, can remarkable increase the absorption of the paeoniflorin in ileum (P < 0.05). After administer the extractive Radix Paeoniae Alba for 5 days, the extraction of Rho123 is significantly increase in ileum (P < 0.01). CONCLUSION: The intestinal absorption of paeoniflorin is zero order rate process and passive absorption. Paeoniflorin is a substrate of P-glycoprotein, and extractive Radix Paeoniae Alba could induce the expression of the P-glycoprotein.

J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Jul 17; Deng P, Chen X, Tang Y, Wang Y, Zhang H, Zhong DArotinoid acid (Ro 13-7410) is the third generation of synthetic retinoid, which was developed for the treatment of psoriasis and other hyperkeratotic skin disorders. The therapeutically active dose is less than 0.5mug/kg body weight/day. To investigate the pharmacokinetics of arotinoid acid, a sensitive and selective liquid chromatographic-tandem mass spectrometric method (LC-MS/MS) for the determination of arotinoid acid in human plasma was developed and validated. The sample processing was carried out in the dark to minimize photodegradation of the analytes. Arotinoid acid and the internal standard (IS), acitretin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was achieved on a Zorbax Extend C(18) column (150mmx4.6mm, i.d., 5mum) using methanol:acetonitrile:5mM ammonium acetate (48:32:20, v/v/v) as the mobile phase at a flow rate of 0.8mL/min. The detection was carried out in multiple reaction monitoring (MRM) mode via negative electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) achieved was 37.1pg/mL with intra-day and inter-day precision (RSD) of 8.7% and 8.5%, and accuracy (RE) of 2.0%. Inter-day and intra-day RSD for three quality control levels (QCs) across validation runs were less than 11.0% and the accuracy ranged from 1.9% to 3.3%. The validated LC-MS/MS method was applied to a phase I clinical pharmacokinetic study after a single oral administration of 40mug arotinoid trometamol to healthy subjects.

J Alzheimers Dis. 2009 Jul 20; Hou Y, Yu YB, Liu G, Luo YSubstantial evidence supports a central role of Abeta in the pathogenesis of Alzheimer's disease (AD). We have demonstrated that FLZ, a synthetic cyclic analogue of natural squamosamide, exhibits neuroprotective actions in cells and mouse models, suggesting future investigation of FLZ as a candidate compound for the treatment of AD. In this study, we found that production of amyloid-beta (Abeta) was reduced by FLZ in Abeta-expressing neuroblastoma cells, which correlates with an increase in the soluble alpha-secretase derived fragment of the amyloid-beta protein precursor (sAbetaPPalpha) in the medium. Moreover, the active form of ADAM10 and AbetaPP were elevated at the cell surface of FLZ-treated cells, consistent with an enhanced co-localization of ADAM10 and AbetaPP on the membrane. Pretreatment with brefeldin, a protein trafficking inhibitor, blocked FLZ-induced translocation of ADAM10 to the cell surface and release of sAbetaPPalpha to the culture medium. Furthermore, oral administration of FLZ to APPswe/PS1 transgenic mice significantly reduced the levels of Abeta, parallel with activation of ADAM10, in the hippocampus. In silico prediction indicates that the structure of FLZ agree with the drug-like rules for absorption and permeability. These findings suggest that reducing Abeta production by FLZ may be mediated by its promotion of AbetaPP non-amyloidogenic alpha-secretase processing, and FLZ has therapeutic potential for the treatment of AD.

Zhongguo Zhong Yao Za Zhi. 2009 Mar; 34(6): 766-9Yan H, He W, Li X, Nie C, Wang L, Li X, Wu L, Tu POBJECTIVE: To establish a HPLC method to determine the carnosic acid in the stomach and intestine of rats and study its tissue distribution characteristics. METHOD: After intragastric administration of carnosic acid (90 mg x kg(-1)), rats for each time-point were sacrificed by decapitation. After removal of the blood, various tissues were rapidly removed and weighted, all tissues were treated with a series of pretreatment before HPLC. Chromatographic separation was achieved on a Kromasil C18 column (4.6 mm x 150 mm, 5 microm) protected by an ODS guard column at 25 degrees C, using acetonitrile-0.1% phosphoric acid solution (55:45) as mobile phase, at a flow rate of 1 mL x min(-1). The wavelength of the UV detector was set at 210 nm for carnosic acid and internal standard. RESULT: Good linearities were obtained in every tissue over a range of 0.3212-160.6 mg x L(-1). The recovery, intra-day and inter-day precision and accuracy of three concentrations of carnosic acid in tissues met the requirements of methodology. And the stability of the tissue samples were also validated. The results of distribution in stomach and intestine showed that the highest concentration was (307.1 +/- 119.2) microg x g(-1) in stomach and (33.32 +/- 17.70) microg x g(-1) in intestine after intragastric administration of carnosic acid. CONCLUSION: The HPLC method was established to determine the concentration of carnosic acid in tissues. This method is quick, precise, and reproducible. It is the first time to study the tissue distribution of carnosic acid in rats after intragastric administration.

Cancer Biol Ther. 2009 Sep 6; 8(17): Sun QM, Miao ZH, Lin LP, Gui M, Zhu CH, Xie H, Duan WH, Ding JAberrant activation of the epidermal growth factor receptor (EGFR) is closely associated with malignant progression of tumors. EGFR inhibitors have been used successfully in clinic in the treatment of solid tumors. In the present study, we revealed that BB, a new synthetic quinonazoline derivative, was a potent EGFR inhibitor. BB selectively inhibited EGFR with a IC(50) value of 50 +/- 37 nM, at least 32-fold more potent than suppressed all other 10 tested receptor tyrosine kinases including the same family member ErbB2 (IC(50) = 5.6 +/- 3.2 muM). BB effectively abrogated autophosphorylation of the EGF-stimulated EGFR and phosphorylation of its key downstream signaling molecules ERK and AKT in A549 cells. BB was shown to suppress EGF-stimulated proliferation of A549 cells with an apparently lower IC(50) value (0.33 +/- 0.07 muM) than that (2.7 +/- 0.4 muM) for the serum-stimulated cells. BB also inhibited the EGF-independent proliferation of a panel of tumor cells. In addition, BB exhibited anti-angiogenesis activity, as evidenced by antagonizing EGF-induced HMEC-1 migration in vitro, blocking HMEC-1 tube formation, and inhibiting microvessel sprouting from rat aortic rings. Most importantly, BB prominently inhibited in vivo tumorigenesis of NIH3T3 cells specifically driven by the activation-mutated EGFR genes. As reported, normal NIH3T3 cells lack tumorigenicity in nude mice. NIH3T3 cells transfected with the EGFR gene with activating mutation (A750P or L858R) produced rapidly growing xenografts in nude mice. BB, when given orally at 100 mg/kg consecutively for 2 weeks, prominently inhibited the growth of the xenografts and reduced the number of microvessels. Taken together, the data indicate that BB is a new selective EGFR inhibitor with potent antitumor activity, revealing its potential as a promising anticancer candidate.

Yao Xue Xue Bao. 2009 May; 44(5): 449-55Li XY, Chen XGProtein kinase C beta (PKCbeta) is a multifunctional serine/threonine protein kinase, which plays an important role in many cell signaling pathways. PKCbeta takes part in multiple physiological processes, including regulation of the cell cycle, differentiation, proliferation, apoptosis and angiogenesis. Increased PKCbeta activity has been observed in many human cancers, such as colon, breast and haematological malignancies. At present, Enzastaurin is mostly studied in preclinical and clinical studies, which is a selective PKCbeta inhibitor. This review focuses on the functional properties of PKCbeta, its role played in tumors and Enzastaurin.

Yao Xue Xue Bao. 2009 May; 44(5): 443-8Ying J, Lü Y, Du GHAmorphous is a special physical state of solid compounds that the positions of the molecules or atoms have no long-range order. Sometimes amorphous compounds have better bioavailability, or achieve ultra-fast absorption in situation of acute and intermittent symptoms than that of morphous compounds, thus change drug efficacy. Besides, different pharmaceutical preparing methods can lead to different characters. Research of amorphous compounds has been a hotspot, both in research field and industry world. However, there are challenges as amorphous compounds could be unstable; unexpected adverse drug reactions may also exist. In this review, recent progress in the research of amorphous pharmaceutical compounds both in the research field and the pharmaceutical industry is reviewed. Factors which can influence the efficacy of amorphous pharmaceuticals are summarized. The prospect of amorphous techniques is also discussed.

Microsc Res Tech. 2009 Jul 20; Choy SK, Tong CS, Zhao ZZThis article presents a novel and effective multistage system for classifying Chinese Materia Medica microscopic starch grain images. The proposed classification system is constructed based on the Gaussian mixture model-based clustering, the feature assignment algorithm, and the similarity measurement. Several features for each starch grain image are extracted and every class of drug is represented by a set of characteristic features. For each stage of the system, only one feature is chosen and assigned to that stage via the feature assignment algorithm, and the corresponding characteristic features are subdivided into smaller subsets based on clustering techniques. At the final stage, each subset contains a certain class of drugs (with corresponding characteristic features) and similarity measurement is carried out for starch grain classification. Three sets of the current state-of-the-art starch grain features including the granulometric size distribution, the chord length distribution, and the wavelet signature are used to construct the system. Experimental results on a database of 240 images of 24 classes of drugs reveal the superior performance of the multistage system. Comparison with the traditional starch grain classification approaches indicates that our proposed multistage method produces a marked improvement in classification performance. Microsc. Res. Tech. 2009. (c) 2009 Wiley-Liss, Inc.

J Cell Biochem. 2009 Jul 21; Ji L, Liu T, Chen Y, Wang ZPyrrolizidine alkaloid (PA) clivorine, isolated from traditional Chinese medicinal plant Ligularia hodgsonii Hook, has been shown to induce apoptosis in hepatocytes via mitochondrial-mediated apoptotic pathway in our previous research. The present study was designed to observe the protection of N-acetyl-cysteine (NAC) on clivorine-induced hepatocytes apoptosis. Our results showed that 5 mM NAC significantly reversed clivorine-induced cytotoxicity via MTT and Trypan Blue staining assay. DNA apoptotic fragmentation analysis and Western-blot results showed that NAC decreased clivorine-induced apoptotic DNA ladder and caspase-3 activation. Further results showed that NAC inhibited clivorine-induced Bcl-xL decrease, mitochondrial cytochrome c release and caspase-9 activation. Intracellular glutathione (GSH) is an important ubiquitous redox-active reducing sulfhydryl (--SH) tripeptide, and our results showed that clivorine (50 microM) decreased cellular GSH amounts and the ratio of GSH/GSSG in the time-dependent manner, while 5 mM NAC obviously reversed this depletion. Further results showed that GSH synthesis inhibitor BSO augmented clivorine-induced cytotoxicity, while exogenous GSH reversed its cytotoxicity on hepatocytes. Clivorine (50 microM) significantly induced cellular reactive oxygen species (ROS) generation. Further results showed that 50 microM Clivorine decreased glutathione peroxidase (GPx) activity and increased glutathione S transferase (GST) activity, which are both GSH-related antioxidant enzymes. Thioredoxin-1 (Trx) is also a ubiquitous redox-active reducing (--SH) protein, and clivorine (50 microM) decreased cellular expression of Trx in a time-dependent manner, while 5 mM NAC reversed this decrease. Taken together, our results demonstrate that the protection of NAC is major via maintaining cellular reduced environment and thus prevents clivorine-induced mitochondrial-mediated hepatocytes apoptosis. J. Cell. Biochem. (c) 2009 Wiley-Liss, Inc.

J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Jun 10; Zhang H, Zhong D, Zhang Z, Dai X, Chen XA sensitive and rapid liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed and validated for the determination of deserpidine in human plasma. The plasma samples were prepared using liquid-liquid extraction (LLE) with ethyl ether-dichloromethane (3:2, v/v). Chromatographic separation was accomplished on an Ultimate XB-C18 column. The mobile phase consisted of methanol-5mM ammonium acetate-formic acid (72:28:0.036, v/v/v). Detection of deserpidine and the internal standard tropisetron was achieved by tandem mass spectrometry with an electrospray ionization interface in positive ion mode. The lower limit of quantification was 4.0pg/ml. The linear range of the method was from 4.0 to 2000pg/ml. The intra- and inter-day precisions were lower than 14.7% in terms of relative standard deviation (RSD), and the accuracy was within +/-8.7% in terms of relative error (RE). This validated method was successfully applied for the evaluation of pharmacokinetics of deserpidine after a single oral administration dose of 0.25mg deserpidine to 22 healthy volunteers.

Yao Xue Xue Bao. 2009 May; 44(5): 456-61Su FQ, Li HY, Zhang Y, Hou SJ, Lei PS, Chen XGThis study is to investigate the effect of Icogenin on and its mechanism in anti-metastasis of pancreatic cancer BxPC3 cells in vitro. Using transwell assay, the effects of Icogenin on the invasion of BxPC3 cells were measured. The abilities of cell motility and adhesion in BxPC3 cells were detected by MTT assay and wound healing assay, respectively. The MAPK signal pathway protein expressions were analyzed with Western blotting. Also, the activity of MMP2 was observed by zymography assay. Icogenin inhibited the abilities of motility, adhesion and invasion of pancreatic cancer BxPC3 cells in vitro (P < 0.05), in a dose-depended manner, and inhibited the secretion of MMP2 and phosphorylation of ERK. PD98059 and U0126 which were ERK inhibitors could suppress the abilities of invasion and metastasis of pancreatic cancer BxPC3 cells. It is concluded that Icogenin can inhibit the abilities of invasion and metastasis of pancreatic cancer in vitro by inhibiting the secretion of MMP2 and phosphorylation of ERK.

Yao Xue Xue Bao. 2009 May; 44(5): 500-5Wang JB, Jin C, Li HF, Li HB, Xiao XH, Qu YThe essential issues of bioassay methods for quality control of traditional Chinese medicines (TCM) were investigated and discussed through the instantiation of developing the bioassay methods for laxative drugs. For the relatively broad variation of the quality of TCM, which might be influenced much by many factors, the parallel lines model of quantitative response is preferred to control the quality of TCM for its relatively high accuracy. The parallel model of quantal response is alternative while the sample can not meet the reliability standard for quantitative response model. According to the requirement of homogeneity between reference and sample for bioassay, the extract from referenced crude meterial is suitable to be used as reference substance after standardizing and defining by chemical substances, and to give reference to the establishment and reproducibility of authorized standard substance. The results of determination of the purgative biopotency of different species of rhubarb and the compound preparations showed that the bioassay methods and self-made reference substance established in this study could be used to control the quality of laxative medicines.

PLoS One. 2009; 4(7): e6304Zhang J, Li C, Shi T, Chen K, Shen X, Jiang HGlucokinase (GK), a glucose sensor, maintains plasma glucose homeostasis via phosphorylation of glucose and is a potential therapeutic target for treating maturity-onset diabetes of the young (MODY) and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). To characterize the catalytic mechanism of glucose phosphorylation by GK, we combined molecular modeling, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) calculations, experimental mutagenesis and enzymatic kinetic analysis on both wild-type and mutated GK. Our three-dimensional (3D) model of the GK-Mg(2+)-ATP-glucose (GMAG) complex, is in agreement with a large number of mutagenesis data, and elucidates atomic information of the catalytic site in GK for glucose phosphorylation. A 10-ns MD simulation of the GMAG complex revealed that Lys169 plays a dominant role in glucose phosphorylation. This prediction was verified by experimental mutagenesis of GK (K169A) and enzymatic kinetic analyses of glucose phosphorylation. QM/MM calculations were further used to study the role of Lys169 in the catalytic mechanism of the glucose phosphorylation and we found that Lys169 enhances the binding of GK with both ATP and glucose by serving as a bridge between ATP and glucose. More importantly, Lys169 directly participates in the glucose phosphorylation as a general acid catalyst. Our findings provide mechanistic details of glucose phorphorylation catalyzed by GK, and are important for understanding the pathogenic mechanism of MODY.

Evid Based Complement Alternat Med. 2009 Jul 16; Khuda-Bukhsh AR, Bhattacharyya SS, Paul S, Dutta S, Boujedaini N, Belon PIn homeopathy, ability of ultra-high diluted drugs at or above potency 12C (diluted beyond Avogadro's limit) in ameliorating/curing various diseases is often questioned, particularly because the mechanism of action is not precisely known. We tested the hypothesis if suitable modulations of signal proteins could be one of the possible pathways of action of a highly diluted homeopathic drug, Secale cornutum 30C (diluted 10(60) times; Sec cor 30). It could successfully combat DMBA + croton oil-induced skin papilloma in mice as evidenced by histological, cytogenetical, immunofluorescence, ELISA and immunoblot findings. Critical analysis of several signal proteins like AhR, PCNA, Akt, Bcl-2, Bcl-xL, NF-kB and IL-6 and of pro-apoptotic proteins like cytochrome c, Bax, Bad, Apaf, caspase-3 and -9 revealed that Sec cor 30 suitably modulated their expression levels along with amelioration of skin papilloma. FACS data also suggested an increase of cell population at S and G2 phases and decrease in sub-G1 and G1 phages in carcinogen-treated drug-unfed mice, but these were found to be near normal in the Sec cor 30-fed mice. There was reduction in genotoxic and DNA damages in bone marrow cells of Sec Cor 30-fed mice, as revealed from cytogenetic and Comet assays. Changes in histological features of skin papilloma were noted. Immunofluorescence studies of AhR and PCNA also suggested reduced expression of these proteins in Sec cor 30-fed mice, thereby showing its anti-cancer potentials against skin papilloma. Furthermore, this study also supports the hypothesis that potentized homeopathic drugs act at gene regulatory level.

Inflammation. 2009 Jul 16; An Y, Liu K, Zhou Y, Liu BAntidiabetic effects of salicylates have been known for years, however the cellular and molecular mechanisms of the hypoglycemic activity are not well elucidated. We examined the effects of salicylate on inflammation-related changes in gene or/and protein expressions of several adipokines in 3T3-L1 adipocytes and of LPS-induced inflammatory factors in RAW 264.7 cell. Especially, we focused our attention on the cross-talk between the macrophages and adipocytes. Exposure to RAW-CM medium resulted in an increase in the gene expression or/and protein secretion of TNF-alpha, IL-6 and resistin, and at the same time, a decrease in the gene expression of PPARgamma and adiponectin in 3T3-L1 adipocytes. Salicylate effectively reversed these changes, and up-regulated glucose consumption in adipocytes. We also found salicylate inhibited phosphorylation of NF-kappaB in RAW-CM-stimulated adipocytes. We conclude salicylate blocks inflammatory process in the pathogenesis of inflammation-related insulin resistance.

J Pharm Biomed Anal. 2009 May 30; Chen D, Wang J, Jiang Y, Zhou T, Fan G, Wu YThe pressurized capillary electrochromatography (pCEC) was utilized for the separation and determination of coumarins in Fructus cnidii extracts from 12 different regions. After a thorough study of analytical parameters such as acetonitrile content of the mobile phase, the concentration and pH of the buffer, and the applied voltage, a methodology was proposed to separate and determine six coumarins of F. cnidii extracts in less than 15min. The experiments were performed in an in-house packed column with a monolithic outlet frit under the optimal conditions: pH 4.0 ammonium acetate buffer at 10mM containing 50% acetonitrile at -6kV applied voltage. The calibration curves were linear in the range of 10.0-100.0mug/mL for bergapten, 20.0-200.0mug/mL for imperatorin, 5.0-400.0mug/mL for osthole, 10.0-100.0mug/mL for 2'-acetylangelicin, 10.0-200.0mug/mL for oroselone, and 10.0-200.0mug/mL for O-acetylcolumbianetin. The correlation coefficients were between 0.9967 and 0.9995. With this pCEC system, fingerprints of F. cnidii extracts were preliminarily established to distinguish three types of coumarins by characteristic peaks, and the quality of various sources of raw materials was evaluated by determining the contents of six coumarins.

Planta Med. 2009 Jul 13; Chen L, Xuan LJ, Wang YPMagnesium lithospermate B (MLB), sodium rosmarinate (SR), and magnesium lithospermate (ML) are biologically active components isolated from aqueous extracts of the Chinese medicine Danshen, the dried root and rhizome of SALVIA MILTIORRHIZA Bunge (Labiatae). These compounds share similar chemical structures of polyphenols and oligomer condensates with caffeic acid. In this study, we compared the effects of MLB, ML, and SR on intracellular Ca (2+) concentrations ([Ca (2+)] (i)) in cultured rat thoracic aorta vascular smooth muscle cells (VSMCs), using the Ca (2+)-sensitive dye, Fluo-3, as an indicator. In our experiments, MLB, ML, and SR did not affect the basal value of [Ca (2+)] (i) in VSMCs. In the absence of extracellular Ca (2+), the [Ca (2+)] (i) increase in VSMCs induced by 20 microM ATP was attenuated by MLB, ML, and SR in a dose-dependent manner. Moreover, under this condition, MLB suppressed the increase in [Ca (2+)] (i) in VSMCs induced by 1 microM thapsigargin, but not ML or SR. In the presence of extracellular Ca (2+), the [Ca (2+)] (i) increase in VSMCs induced by 60 mM KCl was attenuated by MLB and SR in a dose-dependent manner, but not by ML. These results suggest that MLB, ML and SR regulate Ca (2+) homeostasis in VSMCs via different pathways. Our findings should aid in elucidating the mechanisms of the vasodilator action of aqueous extracts of SALVIA MILTIORRHIZA.

Protein Expr Purif. 2009 Jul 3; Guo C, Lian Y, Liu Q, Liu J, Zhang Y, Lin DMouse lipocalin6 (mLcn6) was recently identified to be specifically expressed in the epididymis and speculated to may play a role in sperm maturation. However, further studies were hindered due to the bottleneck to obtain enough recombinant mLcn6 proteins. In this article, GB1 tag was successfully applied to improve the soluble expression of mLcn6. Thermal unfolding experiments demonstrate that GB1 can enhance the structural stability of mLcn6. Fluorescence spectroscopy experiments show that mLcn6 prepared according to our procedure has high affinities to both retinoic acid (K(d) = 810 nM) and retinol (K(d) = 210 nM). In conclusion, soluble, stable and active mLcn6 was recombinantly prepared with the help of the GB1 tag, which will facilitate the structural and functional studies of mLcn6.

J Phys Chem B. 2009 Jul 7; Shen J, Li W, Liu G, Tang Y, Jiang HEstrogen receptors (ER) belong to the nuclear receptor superfamily, and two subtypes, ERalpha and ERbeta, have been identified to date. The differentiated functions and receptor expressions of ERalpha and ERbeta made it attracted to discover subtype-specified ligands with high selectivity. However, these two subtypes are highly homologous and only two residues differ in the ligand binding pocket. Therefore, the mechanism of ligand selectivity has become an important issue in searching selective ligands of ER subtypes. In this study, steered molecular dynamics simulations were carried out to investigate the unbinding pathways of two selective ERbeta ligands from the binding pocket of both ERalpha and ERbeta, which demonstrated that the pathway between the H11 helix and the H7 approximately H8 loop was the most probable for ligand escaping. Then potentials of mean force for ligands unbinding along this pathway were calculated in order to gain insights into the molecular basis for energetics of ligand unbinding and find clues of ligand selectivity. The results indicated that His524/475 in ERalpha/ERbeta acted as a "gatekeeper" during the ligand unbinding. Especially, the H7 approximately H8 loop of ERbeta acted as a polar "transmitter" that controlled the ligand unbinding from the binding site and contributed to the ligand selectivity. Finally, the mechanism of ligand selectivity of ER subtypes was discussed from a kinetic perspective and suggestions for improving the ligand selectivity of ERbeta were also presented. These findings could be helpful for rational design of highly selective ERbeta ligands.

BJOG. 2009 Jul 7; Witt A, Kaufmann U, Bitschnau M, Tempfer C, Ozbal A, Haytouglu E, Gregor H, Kiss HPlease cite this paper as: Witt A, Kaufmann U, Bitschnau M, Tempfer C, Ozbal A, Haytouglu E, Gregor H, Kiss H. Monthly itraconazole versus classic homeopathy for the treatment of recurrent vulvovaginal candidiasis: a randomised trial. BJOG 2009; DOI: 10.1111/j.1471-0528.2009.02262.x.Objective Antimycotics effectively treat sporadic and recurrent vulvovaginal candidiasis (RVVC). Classic homeopathy (CH) is also used to treat this condition. We compared the efficacy of CH and itraconazole in reducing the frequency of RVVC episodes. Design Single-centre, prospective, randomised trial. Sample One hundred-and-fifty patients with a history of RVVC and an acute episode of VVC. Methods Women were randomised into 3 groups: itraconazole with lactobacilli (group 1), itraconazole without lactobacilli (group 2) and CH (group 3). Itraconazole treatment of acute infection was followed by a 6-month maintenance regimen with monthly single-day itraconazole (200 mg bid). Women in group 1 were given additional vaginal lactobacilli for 6 days per month throughout the maintenance regimen Thereafter, patients were followed without treatment for 6 months. CH treatment was performed for 12 months. Results Women in groups 1 and 2 reached a culture-free status significantly earlier than women in group 3 (log-rank test; P < 0.0001). Specifically, before the start of the maintenance regimen, 44 of 49 women (89.8%) in group 1 and 40 of 47 women (85%) in group 2 were free of Candida detectable by culture, 22 of 46 (47%) women in group 3 reached a culture-free status after the first visit, but had a recurrence significantly earlier compared with women in groups 1 and 2 (log-rank test; P = 0.002). After 12 months, 19 of 25 (76%) women in group 1, 18 of 23 (78%) women in group 2 and 9 of 23 (39%) women in group 3 were free of culture-detectable Candida. Assessment of RVVC-associated complaints by VAS score showed that women in group 3 had a significantly higher level of discomfort (36.8, 25.1 and 27.7 respectively; P < 0.001) and were significantly less satisfied (59.2, 68.2 and 71.7 respectively; P < 0.001) than patients in groups 1 and 2. Conclusions Monthly cycle-dependent itraconazole is more effective than CH in the treatment of RVVC. Lactobacilli do not confer an added benefit.

Rev Hist Pharm (Paris). 2009 Feb; 56(360): 469-82Pinet PMen were very early fascinated by magnetism because of its manifest and particular working at distance, which looked different of gravity. It was tryed to be explained by mecanism, for exemple Descartes and Boyle. Paracelse valued the therapeutics with magnets and conceived medicines as working by a magnetic virtue. Gilbert limited the medicinal properties of magnet but helded it to be animated. Many authors praised remedies that work at distance of the evil as Bacon, Van Helmont, Croll, Porta, Goclenius, Digby. Such a belief related to magic ideas of this time. In the Bacon's way Boyle collected facts of magnetic cures, and his actual testing of the divisibility of bodies led him to conceive imponderable corpuscles. Newton supposed a subtil and universel fluid going through every solid body. Mesmer misappropriated this idea by founding the animal magnetism of which physical working was only proceeding from the inside of the patient by an effect of suggestion (psychosomatic). Homeopathy took again the notion of remedies having an infinite or a magnetic virtue, which partly issued from Paracelse's and Mesmer's doctrines, which were extolled in Germany at the time of Hahnemann. The latter decided in favour of a spiritualist and not corpuscular interpretation of the working of his homeopathic medicines.

J Org Chem. 2009 Jul 2; Huang H, Jiang H, Chen K, Liu HWe developed a simple and convenient copper-catalyzed method for the synthesis of quinoline-2-carboxylate derivatives through sequential intermolecular addition of alkynes onto imines and subsequent intramolecular ring closure by arylation. The efficiency of this system allowed the reactions to be carried out at room temperature.

Acta Pharmacol Sin. 2009 Jul 6; Shi YF, Zhang HY, Wang W, Fu Y, Xia Y, Tang XC, Bai DL, He XCAim:To design novel bifunctional derivatives of huperzine B (HupB) based on the concept of dual binding site of acetylcholinesterase (AChE) and evaluate their pharmacological activities for seeking new drug candidates against Alzheimer's disease (AD).Methods:Novel 16-substituted bifunctional derivatives of HupB were synthesized through chemical reactions. The inhibitory activities of the derivatives toward AChE and butyrylcholinesterase (BuChE) were determined in vitro by modified Ellman's method. Cell viability was quantified by the reduction of MTT.Results:A new preparative method was developed for the generation of 16-substituted derivatives of HupB, and pharmacological trials indicated that the derivatives were multifunctional cholinesterase inhibitors targeting both AChE and BuChE. Among the derivatives tested, 9c, 9e, 9f, and 9i were 480 to 1360 times more potent as AChE inhibitors and 370 to 1560 times more potent as BuChE inhibitors than the parent HupB. Further preliminary pharmacological trials of derivatives 9c and 9i were performed, including examining the mechanism of AChE inhibition, the substrate kinetics of the enzyme inhibition, and protection against hydrogen peroxide (H(2)O(2))-induced cytotoxicity in PC12 cells.Conclusion:Preliminary pharmacological evaluation indicated that 16-substituted derivatives of HupB, particularly 9c and 9i, would be potentially valuable new drug candidates for AD therapy, and further exploration is needed to evaluate their pharmacological and clinical efficacies.Acta Pharmacologica Sinica advance online publication, 6 July 2009; doi: 10.1038/aps.2009.91.

J Nat Prod. 2009 Jul 2; Zhang GP, Xiao ZY, Rafique J, Arfan M, Smith PJ, Lategan CA, Hu LHSix new prenylated isoflavanones named sophoronols A-F (1-6), together with eight phenolic constituents, were isolated from the roots of Sophora mollis. Their structures and stereochemistry were established by 1D and 2D NMR techniques, especially HMBC and NOESY as well as CD results. Componds 3 and 5 exhibited moderate anitplasmodial activity against the CQS D10 strain of Plasmodium falciparum, with IC(50) values of 12.9 and 12.8 muM, respectively.

Acta Pharmacol Sin. 2009 Jul; 30(7): 879-888Wang J, Zhang HY, Tang XCVascular dementia (VaD) is a progressive neurodegenerative disease with a high prevalence. Several studies have recently reported that VaD patients present cholinergic deficits in the brain and cerebrospinal fluid (CSF) that may be closely related to the pathophysiology of cognitive impairment. Moreover, cholinergic therapies have shown promising effects on cognitive improvement in VaD patients. The precise mechanisms of these cholinergic agents are currently not fully understood; however, accumulating evidence indicates that these drugs may act through the cholinergic anti-inflammatory pathway, in which the efferent vagus nerve signals suppress pro-inflammatory cytokine release and inhibit inflammation, although regulation of oxidative stress and energy metabolism, alleviation of apoptosis may also be involved. In this paper, we provide a brief overview of the cholinergic treatment strategy for VaD and its relevant mechanisms of anti-inflammation.Acta Pharmacologica Sinica (2009) 30: 879-888; doi: 10.1038/aps.2009.82.

Bioorg Med Chem. 2009 Jun 12; Ye YL, Zhou Z, Zou HJ, Shen Y, Xu TF, Tang J, Yin HZ, Chen ML, Leng Y, Shen JHPPARgamma and 11beta-HSD1 are attractive therapeutic targets for type 2 diabetes. However, PPARgamma agonists induce adipogenesis, which causes the side effect of weight gain, whereas 11beta-HSD1 inhibitors prevent adipogenesis and may be beneficial for the treatment of obesity in diabetic patients. For the first time, we designed, synthesized a series of alpha-aryloxy-alpha-methylhydrocinnamic acids as dual functional agents which activate PPARgamma and inhibit 11beta-HSD1 simultaneously. The compound 11e exhibited the most potent inhibitory activity compared to that of the lead compound 2, with PPARgamma (EC(50)=6.76muM) and 11beta-HSD1 (IC(50)=0.76muM) in vitro. Molecular modeling study for compound 11e was also presented. Compound 11e showed excellent efficacy for lowering glucose, triglycerides, body fat, in well established mice and rats models of diabetes and obesity and had a favorable ADME profile.

Acta Pharmacol Sin. 2009 Jul; 30(7): 889-898Xie KQ, Zhang LM, Cao Y, Zhu J, Feng LYAbstractAim:To understand the mechanism of the transactivation of the epidermal growth factor receptor (EGFR) mediated by the adenosine A(1) receptor (A(1)R).Methods:Primary cultured rat cortical neurons subjected to oxygen-glucose deprivation (OGD) and HEK293/A(1)R cells were treated with the A(1)R-specific agonist N(6)-cyclopentyladenosine (CPA). Phospho-EGFR, Akt, and ERK1/2 were observed by Western blot. An interaction between EGFR and A(1)R was detected using immunoprecipitation and immunocytochemistry.Results:The A(1)R agonist CPA causes protein kinase B (Akt) activation and protects primary cortical neurons from oxygen-glucose deprivation (OGD) insult. A(1)R and EGFR co-localize in the membranes of neurons and form an immunocomplex. A(1)R stimulation induces significant EGFR phosphorylation via a PI3K and Src kinase signaling pathway; this stimulation provides a neuroprotective effect in cortical neurons. CPA leads to sustained phosphorylation of extracellularly regulated kinases 1 and 2 (ERK1/2) in cortical neurons, but only to transient phosphorylation in HEK 293/A(1)R cells. The response to the A(1)R agonist is mediated primarily through EGFR transactivation that is dependent on pertussis toxin (PTX)-sensitive G(i) protein and metalloproteases in HEK 293/A(1)R.Conclusion:A(1)R-mediated EGFR transactivation confers a neuroprotective effect in primary cortical neurons. PI3 kinase and Src kinase play pivotal roles in this response.Acta Pharmacologica Sinica (2009) 30: 889-898; doi: 10.1038/aps.2009.80.

Clin Exp Pharmacol Physiol. 2009 Jun 29; Yang DL, Zhang HG, Xu YL, Gao YH, Yang XJ, Hao XQ, Li XH1. Resveratrol (RSV), a polyphenol in red wine, shows cardioprotective effects in vitro, such as the inhibition of cardiomyocyte hypertrophy induced by angiotensin II or phenylephrine in rat neonatal myocyte cultures, and the suppression of cardiac fibroblast proliferation. This study was aimed to investigate the protective effects of RSV against monocrotaline (MCT)-induced right ventricular hypertrophy in rats. 2. Male Sprague-Dawley rats were given a single injection of MCT (50 mg/kg, sc) and then received treatment with vehicle or RSV (10 and 30 mg/kg, ig, twice daily) for 21 days. Control rats only received normal saline. At the end of treatments, all rats received echocardiography measurements and hemodynamic measurements. The hearts were then subjected to histopathological, untrastructural, and immunohistochemical analysis. 3. MCT produced 33% mortality in model rats, while no mortality occurs when model rats received RSV treatment. MCT increased right ventricular free wall thickness and right ventricular systolic pressure and decreased pulmonary arterial acceleration time at the end of experiment. RSV ameliorated these dynamic changes in a dose-dependent manner. Histologically, MCT produced right ventricular hypertrophy, mitochrondria swollen and cardiomyocyte apoptosis. RSV improved these morphological changes. 4. In conclusion, RSV can inhibit the right ventricular hypertrophy induced by MCT in rats, which is mediated by both direct effect on cardiomyocytes and indirect effect via reducing pulmonary hypertension.

Bioorg Med Chem Lett. 2009 May 15; Zhang X, Zhou Z, Yang H, Chen J, Feng Y, Du L, Leng Y, Shen JSelective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) have considerable potential as treatments for type 2 diabetes. Presented herein are the syntheses, structure-activity relationships, and efficacy evaluation of 4-(phenylsulfonamidomethyl)benzamides as 11beta-HSD1 inhibitors. Through modification of our initial lead 5, we have identified potent and selective 11beta-HSD1 inhibitors, such as 11n, which demonstrated improved glycemic control, decreased serum lipids, and enhanced insulin sensitivity when dosed ip in diabetic ob/ob mice.

Arch Biochem Biophys. 2009 Jun 25; Zhou J, He X, Huang KAccumulating evidence suggests that enhanced peroxynitrite formation occurs during diabetes. This report describes the effect of peroxynitrite on insulin receptor (IR) function. Addition of peroxynitrite to purified IR resulted in concentration-dependent tyrosine nitration and thiol oxidation. Interestingly, the basal and insulin-stimulated IR autophosphorylation and tyrosine kinase activity were upregulated at low peroxynitrite concentrations, but downregulated at high peroxynitrite concentrations. Concomitantly, peroxynitrite dramatically reduced (125)I-insulin binding capacity and phosphotyrosine phosphatase activity of IR preparations. Moreover, SIN-1 administration decreased blood glucose levels in normal mice via upregulation of IR/IRS-1 tyrosine phosphorylation. In contrast, SIN-1 markedly increased blood glucose levels in diabetic mice concomitant with downregulation of IR/IRS-1 tyrosine phosphorylation. Taken together, these data provide new insights regarding how peroxynitrite influences IR function in vitro and in vivo, suggesting that peroxynitrite plays a dual role in regulation of IR autophosphorylation and tyrosine kinase activity, and SIN-1 has hyperglycemic effect in diabetic mice.