Identification and analysis of chemical constituents of Pleione yunnanensis with origin of Pleione yunnanensis / 中国药房

OBJECTIVE To identify an d analyze chemical cons tituents of Pleione yunnanensis with origin of Pleione yunnanensis. METHODS UPLC-Q-Exactive-Plus-Orbitrap-MS was adopted. The determination was performed on Hyperdil GOLD column with mobile phase consisted of 0.1％ formic acid solution-0.1％ formic acid acetonitrile solution （gradient elution ）at the flow rate of 0.3 mL/min. The column temperature was set at 40 ℃，and sample size was 2 µL. The electrospray ion source was adopted，and the scanning range was m/z 100-1 500，and the scanning mode was positive and negative ion exchange mode of full scan+ddMS2. The structure of chemical constituents were determined by using Compound Discoverer 3.1 software，comparing with mzCloud，PubChem network database and OTCML ，on the basis of reference substance and published literatures. RESULTS & CONCLUSIONS A total of 42 chemical constituents were identified （positive ion mode has 24，negative ion mode has 27）， including 13 benzyl succinate glycosides （such as dactylorhin C ，coelovirin A ，militarine），4 phenol glycosides （such as adenosine ， guanosine，gastrodin），3 alkaloids（choline，betaine，berberine），and one flavonoid （nobiletin），7 aromatics（such as DL-lysine ， DL-arginine，DL-glutamine），one sugar （sucrose），3 benzenes（shancigusin H ，shancigusin H isomer ，batatasin Ⅲ）and 10 others （such as p-methoxybenzoic acid ，monomethyl dodecanedioate ，diphenylamine）. Glucose oxybenzyl and some small mole cules are easy to be lost in the cleavage of benzyl succinate glycosides；glycosyl is easy to be lost in the cleavage of phenol glycosides；the cleavage of alkaloids mainly manifest as the cleavage and loss of small molecular substituents ；demethyla- tion reaction is occurred in most flavonoids.

Study on Improvement Effects of Polygonum orientale Flower Extract on Hypoxia-reoxygenation Injury of H 9c2 Cardiomyocytes / 中国药房

OBJECTIVE：To study the improvement effects and mechanism of Polygonum orientale flower extract on hypoxia- reoxygenation injury of H 9c2 cardiomyocytes. METHODS ：H9c2 cardiomyocytes were divided into normal control group ，model group and low- ，medium- and high-concentrations groups of P. orientale flower extract （20，40，80 μg/mL）. Except for normal control group ，other groups were given 800 μmol/L CoCl2 to induce hypoxia-reoxygenation injury model. Cell apoptosis was observed. The levels of Ca 2+（in cytoplasm ），mitochondrial membrane potential （MMP），ATP enzyme （Na+-K+-ATP enzyme ，Ca2+-Mg2+-ATP enzyme） activities， the ratio of cytochrome c （Cyto c ）， protein in cytosol to mitochondria ，phosphorylation levels of reperfusion injury salvage kinase （RISK） signaling pathwayrelated protein [protein kinase B （Akt）and extracellular signal regulated kinase 1/2（ERK1/2）] as well as protein expression of HIF- 1 α were detected respectively. In addition，the cells were divided into normal control group ，model group and P. orientale flower extract group （80 μ g/mL），PI3K inhibitor LY294002+CoCl2 group（15 μmol/L LY294002+80 μmol/L ，LY294002+P. orientale flower extract group （15 μmol/L LY294002+80 μg/mL P. orientale flower extract ），MEK inhibitor PD98059+CoCl2 group（25 μmol/L PD98059+800 μmol/L CoCl2），PD98059+P. orientale flower extract group （25 μmol/L PD98059+80 μg/mL P. orientale flower extract ）. After cultured by the same method ，the phosphorylation levels of Akt protein and ERK1/2 protein in the cells were measured to verify the activation of P. orientale flower extract to RISK signaling pathway. RESULTS：Compared with model group ，nuclear pyknosis and the number of apoptotic bodies were reduced in different concentrations groups of P. orientale flower extract. ROS level ，Ca2+ level（except for low-concentration group ），MMP，ratio of Cyto c in cytoplasm to Cyto c in mitochondria ，protein expression of HIF- 1α were decreased significantly（P＜0.05 or P＜0.01）； the activity of ATP enzyme （except for the low-concentration group ），Akt protein and ERK 1/2 protein phosphorylation level were significantly increased （P＜0.01）. After treated with PI 3K inhibitor LY 294002 and MEK inhibitor PD 98059，Akt protein and ERK 1/2 protein phosphorylation level in cadiomyocyte were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：P. orientale flower extract can improve hypoxia-reoxygenation injury of H 9c2 cardiomyocytes，the mechanism of which may be associated with inhibiting cardiomyocyte apoptosis ，improving ATPase activity ，protecting mitochondria ，regulating RISK signaling pathway related proteins and HIF- 1α protein expression.

Analysis of Chemical Compositions in Miao Medicine Caesalpinia decapetala by UPLC-Q-TOF-MS/MS / 中国药房

OBJECTIVE：To estab lish a method that can comprehensively and rapidly analyze the chemical compositions of Miao medicine Caesalpinia decapetala，and to providing reference for quality control and pharmacodynamic material basis study of C. decapetala . METHODS ：UPLC-Q-TOF-MS/MS was adopted . The determination was performed on Agilent SB-C 18 column with mobile phase consisted of 0.1％ formic acid solution- 0.1％ formic acid acetonitrile （gradient elution ）at the flow rate of 0.25 mL/min. The column temperature was 30 ℃，and sample size was 2 µL. ESI source was applied in negative and positive scanning ion mode and data collection range of m/z 50-1 500. The capillary voltage was 4.5 kV，the atomizing gas （nitrogen）pressure was 1.2 Bar， the solvent removal gas was nitrogen ，the flow rate of solvent removal gas was 8 L/min and the solvent removal gas temperature was 200 ℃. Data Analysis 4.2 software was adopted to analyze fragment ion information of each peak ，and identify chemica l compositions on the basis of relevant literature and mass spectograms of reference substance. RESULTS ：Under positive ion mode ， 9 chemical compounds were identified ；peak 1，2，3，4，5，6，7，8，9 were catechin ，protohematoxylin B ，epicatechin，ethyl gallate，quercetin，luteolin，3-deoxy-hematoxylin chalcone ， isoliquiritigenin and linoleic acid. Under negative ion mode ， U1812403）， totally 21 peaks were confirmed and 13 compounds were identified；peak 3，4，5，6，7，8，9，10，11，12，13，15， 21 were catechins ， brevifolin carboxylic acid ， proto- hematoxylin B ，epicatechin，ethyl gallate ，epicatechin gallate ， quercetin，resveratrol，hematoxylin，luteolin，3-deoxy-hema- toxylin， isoliquiritigenin， linoleic acid. CONCLUSIONS UPLC-Q-TOF-MS/MS method is established successfully for analysis of chemical compositions in C. decapetala .

Study on Metabolism of Miao Medicine Laportea bulbifera Extract in Isolated Human Intestinal Flora / 中国药房

OBJECTIVE：To explore the metabolic charact eristics of Miao medicine Laportea bulbifera extract in isolated human intestinal flora. METHODS ：L. bulbifera was extracted with 70％ ethanol reflux extraction. After concentration，extraction with n-butanol and drying ，L. bulbifera extract was obtained. Taking 0.05 g/mL L. bulbifera extract 1 mL mixed with isolated human intestinal flora fluid 10 mL and cultured for 36 h in anaerobic environment （setting up blank control without drugs or human intestinal bacterial solution ），so as to simulate the metabolic process of the extract in human intestine. The metabolites were detected by UPLC-Q-TOF/MS. The determination was performed on Agilent Eclipse Plus C 18 RRHD column with mobile phase consisted of 0.01％ formic acid water solution- 0.01％ formic acid acetonitrile solution （gradient eluetion ）at the flow rate of 0.25 mL/min. The column temperature was set at 40 ℃，and the sample size was 1 µL. ESI detection was adopted and scanned by negative ion mode （ESI－）；the capillary voltage was 4.5 kV，the ion source temperature was 120 ℃，the collision energy was 15-32 V，and the scanning range was m/z 50-1 000. The “Strip”module of MassLynx V 4.1 software was used to analyze the differential chromatograms between the reaction solution and the blank control of L. bulbifera extract. Mass spectrum data and UNIFI so ftware were used to predict relative molecular weight and formula ；based on the information of substance control and related literature reports ， the structure and biotransformation pathway of L. bulbifera metabolites in isolated human intestinal flora were predicted and analyzed. RESULTS & CONCLUSIONS ： A total of 3 prototype ： products（rutin，quercetin，kaempferol-3-O-rutinoside）and 22metabolites （mainly the metabolites of quercetin ，mono- caffeoylquinic acid ，isoquercitrin，etc.） were detected after metabolized in isolated human intestinal flora. Itsbiotransformation pathway is phase Ⅰ reaction，which mainly consisted of reduction ，oxidation and hydrolysis.

Study on Intestinal Absorption Characteristics of 5 Active Components in Ethanol Extract from Bletilla striata / 中国药房

OBJECTIVE： To investigate absorption kinetic characteristics of main active components as 4-（glucoseoxy）- glucoseoxybenzyl cinnamate （A1）， 2-isobutyl malic acid （A2）， 1，4-bis [4-（glucoxy） benzyl]-2-isobutyl malic acid ester （A3）， dihydrophenanthrenes 1 （A4） and 1，4-bis [4-（glucosoxy） benzyl]-2-isobutyl malic acid ester-2-（4-O-cinnamoyl-6-O-acetyl） glucoside （A5） from ethanol extract of Bletilla striata in the intestines of rats. METHODS： Using puerarin as internal standard， UPLC-MS/MS was used to determined the concentration of A1-A5 in intestinal circulation fluid. The determination was performed on Acquity UPLC BEH C18 column with mobile phase consisted of acetonitrile （containing 0.1％ formic acid）-water （containing 0.1％ formic acid） （gradient elution） at the flow rate of 0.35 mL/min. The column temperature was 45 ℃， and sample size was 3 μL. The positive ion and negative ion scanning were carried out in the multiple reaction monitoring mode by electrospray ion source. The ion pairs for quantitative analysis were m/z 593.2→431.1 （A1）， m/z 189.0→129.0 （A2）， m/z 725.3→457.2 （A3）， m/z 347.1→332.1 （A4）， m/z 1 059.3→793.1 （A5）， m/z 417.0→267.0 （internal standard）. In the in vivo intestinal circulation perfusion model， using accumulative absorption transfer rate （A） and absorption and transformation rate constant （Ka） as indexes， the effects of different doses of ethanol extract from B. striata （low-， medium-， high-dose were 166， 333，667 μg/mL，respectively）， bile， P-glycoprotein （P-gp） inhibitors （verapamil） and different intestinal segments on the absorption of above 5 components were investigated. RESULTS： The linear range of A1， A2， A3， A4 and A5 were 0.22-14.00， 0.34-21.75， 1.99-127.16， 0.15-9.75， 0.16-10.00 μg/mL（r＞0.99）. The limits of quantitation were 0.22， 0.34， 1.99， 0.15， 0.16 μg/mL， respectively. The lowest detection limits were 0.028， 0.085， 0.251， 0.035 and 0.010 μg/mL. RSDs of inter-day and intra-day were all lower than 10％. The recoveries ranged 83.60％-106.91％. Matrix effect did not affect the determination of the substance to be measured. A and Ka values of A1 in B. striata ethanol extract low-dose and medium-dose groups were significantly higher than high-dose group； A value of A3 in low-dose group was significantly higher than medium-dose and high-dose groups （P＜0.05 or P＜0.01）. A and Ka values of A1 and A3 in non-ligation group were significantly lower than control group， while A and Ka values of A4 were significantly higher than control group （P＜0.05 or P＜0.01）. A and Ka values of A1 and A3 in P-gp inhibitor group were significantly lower than control group （P＜0.05 or P＜0.01）. A values of A1 in jejunum group， ileum group and colon group， Ka value of A1 in colon group， A and Ka values of A2 in colon group， A value of A3 in ileum group， A and Ka values of A4 in ileum group and colon group， A values of A5 in jejunum group and ileum group as well as Ka value of A5 in jejunum group were all significantly lower than duodenum group. Ka values of A3 in jejunum group， ileum group and colon group were significantly higher than duodenum group （P＜0.05 or P＜0.01）. CONCLUSIONS： Established UPLC-MS/MS method is specific， sensitive and simple， and it can be used for quantitative analysis and pharmacokinetic study of A1-A5. The 5 active components in B. striata ethanol extract are absorbed by the whole intestine， and the intestinal segments are different. A1 and A3 are absorbed more in intestinal tract and may be saturated. Bile can inhibit intestinal absorption of A1 and A2， but promoted intestinal absorption of A4. A1-A5 may not be the substrate of P-gp.

in vitro Metabolism Pathway of Effective Component of Bletilla striata as Militarine in Liver Microsomes and Kinetics Char- acteristics of Enzyme-catalyzed Reactions / 中国药房

OBJECTIVE： To study in vitro metabolism pathway of effective component of Bletilla striata as Militarine in liver microsomes and kinetics characteristics of enzyme-catalyzed reactions. METHODS： The in vitro incubation system of rat and human liver microsomes was established， and incubation reaction of Militarine was performed. UPLC-QTOF-MS was used to identify the structure of its metabolites in combination with UNIFI database and references. Using puerarin as internal standard， UPLC-Triple Quad-MS was used to quantitatively analyze metabolic transformation of Militarine in rat liver microsomes. The kinetic parameters （vmax， km， CLint） of Militarine enzyme-catalyzed reactions with/without reducing coenzyme Ⅱ （NADPH） were calculated by fitting the curves with GraphPad Prism 5.0 software. RESULTS： After incubation in rat and human liver microsomes， Militarine produced a chemical formula C21H29O11， which was presumed to be a metabolite of Militarine ester bond hydrolysis. The kinetic study of enzyme-catalyzed reactions showed that vmax of Militarine enzyme-catalyzed reactions with/without NADPH were 1.955， 2.129 nmol/（h·mg）； km were 8.601， 9.854 nmol/mL； CLint were 0.227 3， 0.216 1 mL/（h·mg）； there was no significant difference between with NADPH and without NADPH. CONCLUSIONS： The main metabolic pathway of Militarine in liver microsomes is the hydrolysis of C1 and C4 ester bonds. Its metabolism does not depend on the pathway of cytochrome P450 enzymes initiated by NADPH.

Study on Protective Effects of Polygonum orientale Extract on Myocardial Ischemia-reperfusion Injury Model Rats / 中国药房

OBJECTIVE： To study the protective effects of Polygonum orientale extract on myocardial ischemia-reperfusion injury （MIRI） model rats， and to provide reference for it’s deeply development of medicinal source. METHODS： Totally 24 rats were randomly divided into sham operation group （normal saline）， model group （normal saline）， Compound danshen tablet group （positive group， 0.17 g/kg） and P. orientale extract group （86 g/kg， calculated by crude drug）， with 6 rats in each group. All groups were given drugs 2 mL/100 g intragastrically once a day. After 4 d of consecutive administration， MIRI model was induced by the left anterior descending branch of arteria coronaria in all groups except for sham operation group. 24 h after reperfusion， they were given related medicine again. After medication， the changes of electrocardiogram ST segment were monitored in each group. The plasma levels of LDH， CK-MB， CK， cTn-I， SOD and NO were detected in each group. The myocardial infarction rate in each group was calculated and the pathomorphological changes in the myocardium were observed. RESULTS： Compared with sham operation group， ST segment of myocardial electrocardiogram was increased in model group （P＜0.01）. The plasma levels of LDH， CK， CK-MB and cTn-I were increased significantly （P＜0.01）， while the plasma levels of SOD and NO were decreased significantly （P＜0.01）. The rate of myocardial infarction was increased significantly （P＜0.01）， and pathomorphological changes were observed in myocardial tissue such as infiltration of inflammatory cells and loose cytoplasm of cardiac myocytes. Compared with model group， ST segment of myocardial electrocardiogram was decreased significantly in Compound danshen tablet group and P. orientale extract group （P＜0.05）； the plasma levels of LDH， CK， CK-MB and cTn-I were decreased significantly （P＜0.05）， while the plasma levels of SOD and NO were increased significantly （P＜0.05）； the rate of myocardial infarction was decreased significantly （P＜0.05）， and inflammatory cell infiltration and tissue edema in myocardium were relieved to varying degrees. CONCLUSIONS： The protective effect of P. orientale extract protect on MIRI may be exerted by anti-oxidative damage.

Simultaneous determination of seven constituents in Sangqishouwu Tablets by HPLC / 中成药

AIM To establish an HPLC method for the simultaneous content determination of calycosin-7-glucoside,ononin,calycosin,formononetin,oxypaeoniflorin,alibiflorin and benzoylpaeoniflorin in Sangqishouwu Tablets (Talxilli Herba,Astragali Radix,Polygoni multiflori Radix Praeparata,etc.).METHODS The analysis of methanol extract of this drug was performed on a 25 ℃ thermostatic Diamonsil C18 colmn (250 mm × 4.6 mm,5 μm),with the mobile phase comprising of acetonitrile-0.02％ phosphoric acid flowing at 0.9 mL/min in a gradient elution manner,and the detection wavelengths were set at 230 nm and 254 nm.RESULTS Seven constituents showed good linear relationships within their own ranges (r ≥ 0.999 2),whose average recoveries were 97.13％-100.03％ with the RSDs of 0.69％-1.47％.CONCLUSION This sensitive,accurate and specific method can be used for the rapid quality control of Sangqishouwu Tablets.

Study on Quality Standard of Miao Medicine Gaultheria yunnanensis / 中国药房

OBJECTIVE:To establish the quality standard for Gaultheria yunnanensis. METHODS:TLC was adopted for quali-tative identification of samples. Moisture,total ash and acid-insoluble ash were determined. HPLC method was used to determine the content of methyl salicylate. The determination was performed on Eclipse XDB-C18 column with mobile phase consisted of meth-anol-water(62:38,V/V)at the flow rate of 1.0 mL/min. The detection wavelength was set at 307 nm,and column temperature was 30 ℃. The sample size was 10 μL. RESULTS:TLC spots of samples were clear and well separated. The moisture was 8.2%-10.8%,total ash was 0.9%-4.0% and acid-insoluble ash was 0.1%-0.9%. The linear range of methyl salicylate were 0.045-0.73μg(r=0.9999). RSDs of precision,stability and reproducibility tests were no more than 1.0%. The recoveries of meth-yl salicylate were 97.8%-104.3%(RSD=2.6%,n=9). CONCLUSIONS:The established standard can be used for quality control of G. yunnanensis.

In vitro absorption mechanism of Erigeron breviscapus extract in Caco-2 cell monolayer model / 中国药理学通报

Aim To study the absorption mechanism of apigenin-7-O-glucronide, 3,4-Dihydroxycinnamic acid and chlorogenic acid in Erigeron breviscapus extract ( Ebe) by Caco-2 cell monolayer model. Methods The three active ingredients were quantified by UPLC-MS/MS method, and the effect of Ebe concentrations, conveying times, pH values and P-glycoprotein inhibi-tor on the transport of three active ingredients were also investigated. Results Apigenin-7-O-glucronide and chlorogenic acid in Caco-2 cell monolayer model were time-dependent and concentration-dependent. The 3, 4-Dihydroxycinnamic acid in Caco-2 cell uptake was concentration-saturate and P-glycoprotein inhibitor was involved in the uptake process. Conclusion The mechanism of apigenin-7-O-glucronide and chlorogenic acid absorption in cells is mainly through passive trans-port, and the absorption of 3, 4-Dihydroxycinnamic acid is mainly realized by the carrier transport.

Application of an R-group search strategy into three-dimensional quantitative structure-activity relationship of HEA beta-secretase inhibitors and molecular virtual screening / 生物医学工程学杂志

The beta-secretase is one of prospective targets against Alzheimer's disease (AD). A three-dimensional quan titative structure-activity relationship (3D-QSAR) model of Hydroethylamines (HEAs) as beta-secretase inhibitors was established using Topomer CoMFA. The multiple correlation coefficient of fitting, cross validation and external validation were r2 = 0.928, q(loo)2 = 0.605 and r(pred)2 = 0.626, respectively. The 3D-QSAR model was used to search R groups from ZINC database as the source of structural fragments. As a result, a series of R groups with relatively high activity contribution was obtained to design a total of 15 new compounds, with higher activity than that of the template molecule. The molecular docking was employed to study the interaction mode between the new compounds as ligands and beta-secretase as receptors, displaying that hydrogen bond and hydrophobicity played important roles in the binding affinity between the new compounds and beta-secretase. The results showed that Topomer CoMFA and To pomer Search could be effectively used to screen and design new molecules of HEAs as beta-secretase inhibitors, and the designed compounds could provide new candidates for drug design targeting AD.

Oil of Piper longum unsaponifiable matter prevents cholesterol gallstone formation / 药学学报

To observe the effect of various doses of oil of Piper longum unsaponifiable matter (OPUM) to cholesterol gallstones in experimental mice. C57BL/6 mice (n = 60) were randomly divided into 6 groups: control group, model group, OPUM (15, 30 and 60 mg x kg(-1)) group and ursodeoxycholic acid (UDCA, 60 mg x kg(-1)) group, administered for 10 weeks. The level of serum lipid and liver function enzymes were tested. The gallbladder was removed and bile was obtained by centrifugation. Next, the levels of the bile total cholesterol (TC), phospholipid (PL) and bile acid (TBA) were measured. The indicators of lipid peroxidation were determined and cholesterol saturation index (CSI) was calculated. The liver histological changes were observed by HE staining. The results showed that serum TC, TG (triglycerides) and AST (aspartate transaminase) contents, gallbladder cholesterol crystallization and CSI increased significantly (P < 0.05). In addition, the activity of SOD decreased significantly and MDA content increased significantly in liver (P < 0.05). HE staining results showed that the hepatic cord disorder and intracellular lipid droplets increased significantly. All results indicate that lithogenic diet lead to the formation of cholesterol gallstones. In OPUM (30 and 60 mg x kg(-1)) group, serum TC, TG and AST content, gallbladder cholesterol crystallization and CSI decreased significantly, the activity of SOD increased significantly and MDA content decreased significantly. HE staining results showed that OPUM can improve the morphology of liver cell, reduce the degree of hepatic cord disorders and restore the cell morphology close to normal. The cause of OPUM prevents cholesterol gallstone formation maybe due to protect the integrity of the liver cells, lower CSI, and reduce cholesterol crystal formation and hence prevent cholesterol gallstone formation.

Comparative analysis of clinicopathological findings and outcome of Henoch-Schnlein nephritis and IgA nephropathy in adults / 北京大学学报(医学版)

0.05).In patients with HSPN capillary wall staining for IgA was more frequently found than in IgAN(71.0% vs 43.5%,P=0.013).With creatinine level doubling as the end point,the follow-up data indicated that the renal survival was 87.1% in HSPN and 91.9% in IgAN and there was no statistically significant difference between HSPN and IgAN(P=0.481).Conclusion:Although significant pathological difference was found between HSPN and IgAN,the renal clinical manifestations and long term outcome were similar between the two diseases in adults.

The activity of aminopeptidase N and gallstone formation / 中国普通外科杂志

Objective To study the activity of aminopeptidase N (APN) in the bile of patients with cholesterol gallstones (CGS) and rabbits fed with lithogenic diets (LD) to explore the lithogenic role of APN in the formation of gallstones. Methods Enzymatic activity of APN and lipid concentrations in bile of investigated CGS patients and LD fed rabbits were measured. The relationship between the activity of APN and CSI was statistically analyzed.Results APN activity in CGS patients′ gallbladder (GB) and bile duct (BD) biles 〔(66 0?24 1)*!U/ml and (38 4?9 4)*!U/ml〕 as well as in LD fed rabbits′ GB and BD biles 〔(14 5?4 08)*!U/ml and (9 13?1 67)*!U/ml〕 were significantly higher than that in the corresponding biles of their clinical and experimental control (CC and EC) groups 〔(23 0?7 7)*!U/ml〕 in GB bile of CC group and 〔(6 05?0 95)*!U/ml and (3 09?0 92)*!U/ml〕 in EC group. All P values were less than 0 05. A statistical positive correlation was found between the APN and CSI value in each of the groups.Conclusions It is more likely that APN might play certain role in the early neucleating period of the pathogenesis of gallstones based on its proneucleative potential and measurement of APN activity may serve as a predictive marker among patients with high risk of gallstone formation.