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??OBJECTIVE To establish an HPLC-ELSD method for determination of mesaconine.METHODS Gemini C₁₈ 110A column (4.6 mm??150 mm,5 ??m) was adopted with the mobile phase consisting of methanol-0.1%TFA (25:75) at the flow rate of 0.6 mL??min^-1. The detector was Alltech ELSD 2000ES, with the drift tube temperature of 90 ?? and the airflow velocity of 2.3 L??min^-1. RESULTS Under this conditions, mesaconine and the related substances were well separated. The linear range of mesaconine was 0.40-0.60 mg??mL^-1 (r=0.999 6). RSD of injection precision and repeatability test were 0.04% (n=5) and 0.7% (n=6), the intra day and inter day precision RSD were 0.4% and 0.6%, respectively. The average recovery was 101.0% (RSD=1.1%, n=9). CONCLUSION The established method was specific, simple, accurate, and suitable for determination of mesaconine.     

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??OBJECTIVE ??-Conotoxin LtIA (??-CTX LtIA, LtIA) is a specific inhibitor of ??3??2 nicotinic acetylcholine receptors (nAChRs) from Conus litteratus, a marine snail native to Hainan. The aim of this study was to evaluate the analgesic activity of ??-CTX LtIA. METHODS The analgesic effect of ??-CTX LtIA on pain models was evaluated using mice hot-plate and tail-flick models by intracerebroventricular (icv) injection. RESULTS In tail-flick test, the maximum analgesia percentage (PMAP) was 37.74% at 15 min after LtIA administration by icv injection with dose of 0.2 nmol per mouse. While in hot-plate test, PMAP was 48.81% at 60 min after LtIA administration by icv injection with same dose of 0.2 nmol per mouse. ??-CTX LtIA showed good analgesic activity in two pain models. CONCLUSION ??-CTX LtIA exhibits good analgesic activity by specific interaction with ??3??2 nAChRs subtype. These RESULTS have great significance for the research and development of LtIA painkiller in the future.     

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??OBJECTIVE To study the chemical constituents of the chloroform extract from the aerial parts of Artemisa sacrorum. METHODS The chemical constituents were isolated and purified by silica gel and LH-20 column chromatography and preparation HPLC. Their structures were identified by spectral analysis methods. RESULTS Thirteen compounds were obtained and identified as 5-hydroxyl-7,4??-dimethoxyflavone(1), 4-hydroxylacetophenone(2), 5,4??-dihydroxyl-7,3??-dimethoxyflavone(3), 5,7-dihydroxyl-6,4??-dimethoxyflavone(4), 5,7-dihydroxyl-4??-methoxyflavone(5), 5,4??-dihydroxyl-7-methoxyflavone(6), caffeic acid(7), 8-hydroxyl-6,7-dimethoxycoumarin(8), 3,4-dihydroxylbenzoic acid(9), acetophenone-4-O-??-D-glucoside(10), 6-methoxycoumarin-7-O-??-D-glucoside(11), 6,8-dimethoxycoumarin-7-O-??-D-glucoside(12), and 2-hydroxyl-6-methoxyacetophenone-4-O-??-D-glucoside(13). CONCLUSION Compounds 3, 4, 5, 9, 10 and 12 are isolated from this plant for the first time.
     

LC-MS/MS�ⶨ��Ѫ�����������Ũ�ȼ���ҩ��ѧ�о�

??OBJECTIVE To establish an LC-MS/MS method for the determination of pregabalin, which was used subsequently to investigate the pharmacokinetics of pregabalin in healthy Chinese volunteers. METHODS Gabapentin was used as internal standard. The separation was achieved on a Waters Symmetry C₁₈ column (3.9 mm??150 mm,5 ??m) with a mobile phase consisting of acetonitrile -5 mmoL??L^-1 ammonium acetate and 0.3% formic acid aqueous solution (8/92,V/V) at a flow rate of 1.0 mL??min^-1 within 6.0 min. Pregabalin and the internal standard were measured by a triple-quadrupole mass spectrometer in positive electron atmospheric-pressure chemical ionization (APCI) mode using multiple reaction monitoring (MRM). The extracted ions monitored following MRM transitions were m/z 160.1??142.1 for pregabalin and m/z 172.1??154.1 for the internal standard gabapentin. Plasma samples were pretreated by methanol precipitation. RESULTS The calibration curve of pregabalin in human plasma was linear over the concentration range of (30-5 000) ng??mL^-1. The lower limit of quantitation was 30 ng??mL^-1. The intra- and inter-run precisions at three quality control levels were within 2.0%-6.2%, the relative deviation of the assay was within -10.5%-3.0%. The plasma samples were stable at room temperature (25 ??) for 6 h, at -30 ?? for 27 d and during three freeze-thaw cycles. The determination and the result of incurred sample reanalysis met the requirements. CONCLUSION The method is proved to be convenient, accurate and sensitive. The method is proved to be suitable for the pharmacokinetics of pregabalin in healthy Chinese volunteers after a single oral dose of 75 mg pregabalin capsule.     

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??OBJECTIVE To establish an improved LC method combined with pulsed electrochemical detection for the analysis of etimicin sulfate. METHODS The mobile phase was composed of 40 mL of acetonitrile and 960 mL of an aqueous solution containing 15 mL??L^-1 of trifluoroacetic acid,500 ??L??L^-1 of pentafluoropropionic acid, 8 mL??L^-1of sodium hydroxide (50%) and 1.5 g??L^-1of sodium sulfate. The pH of the aqueous solution was adjusted to 3.5 with 50% NaOH solution. A pulsed electrochemical detector, which was kept at 35 ?? in a hot air oven was adopted. The electrochemical cell consisted of a working electrode, a pH-Ag/AgCl reference electrode and a titanium counter electrode. The working electrode was a gold electrode (diameter 3 mm)and a quadruple-potential waveform (QPW) was selected as detection waveform. The 0.8 mol??L^-1 NaOH solution was added post column at a flow rate of 0.4 mL??min^-1. RESULTS In total, 22 impurities could be separated. The LOD and LOQ of etimicin were found to be 2 ng and 6 ng respectively. The linearity of the calibration curve for etimicin ranged from 0.24 to 45 ??g??mL^-1 with a coefficient of correlation equal to 0.999 7. The repeatability RSDs (n=6) of the content and total impurities in one sample were 0.7% and 1.72% respectively. The inter-day repeatability RSDs (n=18) of the content and total impurities in one sample were 0.98% and 1.71% respectively. The sample solution was stable within 12 h. CONCLUSION Compared with previously published methods, this improved method shows higher sensitivity, better separation ability and robustness and has been incorporated by the Chinese Pharmacopoeia (Ch.P.) 2015 for analysis of etimicin sulfate.     

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??OBJECTIVE To establish and compare the HPLC fingerprints of different medicinal parts of Morus alba. METHODS An HPLC analysis was performed on an Agilent Eclipse XDB C₁₈ (4.6 mm??250 mm, 5 ??m) chromatographic column, using gradient elution with acetonitrile and 0.1% aqueous formic acid at a flow rate of 1.0 mL??min^-1. The column temperature was kept at 30 ??, and the detection wavelength was set at 254 nm. The data was analyzed with Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004A). RESULTS The HPLC fingerprints and common models of different medicinal parts of M. alba were established. The numbers of common peaks obtained in the fingerprints of Mori Cortex, Mori Ramulus, Mori folium, and Mori Fructus were 10, 11, 12, and 8, respectively. Ten characteristic peaks were identified by comparison with the reference substances and accurate molecular weights determined by UPLC-Q-TOF/MS. Mori Cortex and Mori Ramulus both had mulberroside A, oxyresveratrol, kuwanon G, and morusin. Rutin and isoquercitrin were detected in both Mori folium and Mori Fructus. CONCLUSION The method is stable, reliable, and repeatable. The composition profiles of different medicinal parts are established, which provides a scientific basis for the quality control of M. alba.     

N,N-���׻�-[N��,N��-˫(Ӳ֬����-1-�һ�)]1,3-�������ĺϳɡ����ʼ���Ϊ�װ����������Ӧ��

??OBJECTIVE To synthesize a novel cationic lipid,N,N-dimethyl-[N??,N??-di-(stearoyl-1-ethyl)] 1,3-diaminopropane (DMSP),and evaluate its feasibility as methotrexate(MTX) carrier. METHODS DMSP and phosphatidylcholine were employed to prepare liposomes by reverse phase evaporation method,and then MTX was entrapped by physical mixing. The entrapment efficiency was determined by ultracentrifugation,and its release ratio was evaluated by dialysis. The morphology of liposomes was observed under transmission electron microscope. The average diameter and Zeta potential were determined by laser particle size analyzer. MTT test was used to evaluate the cytotoxicity of liposomes as drug carrier and the inhibition of cancer cells growth. RESULTS The obtained liposomes showed regular shape and uniform size,with a mean Zeta potential of +(36.26??4.77)mV and average diameter of 120 nm. The liposomes had low hemolytic activity and cytotoxicity. With the help of DMSP the cationic liposomes achieved a very high entrapment efficiency for the hydrophilic drug MTX (91.50??1.02)%. The inhibition of the MTX liposomes on cancer cells growth was much higher than that of MTX solution. CONCLUSION DMSP is a novel cationic lipid with low cytotoxicity and high entrapment efficiency,which has a great application potential in drug delivery system.     

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??OBJECTIVE To develop an high-performance liquid chromatography (HPLC) method with diode array detector (DAD) for determination of acarbose in acarbose tablets. METHODS An Alltima C₁₈ column (4.6 mm??250 mm,5 ??m) was used for the separation, with acetonitrile-10 mmol??L^-1 ammonium dihydrogen phosphate (containing 0.04% sodium 1-octanesulfonate, adjusted pH to 3.3 with H₃PO₄) (15??85) as the mobile phase at the flow rate of 0.8 mL??min^-1. The detection wavelength was set at 200 nm and the column temperature was maintained at 30 ??. RESULTS The method showed good linearity with a correlation coefficients (r) of 1.000 0. The specificity study demonstrated satisfactory resolutions between acarbose, other ingredients in the drug and forced degradation products. The precision and stability were satisfactory with the relative standard deviations (RSDs) of 0.088% and 0.22%, respectively. The average spiked recovery was 98.49% with RSD of 0.24% (n=9). The content of acarbose in four batches of acarbose tablets was 51.69, 51.75, 51.72 and 51.62 mg??tablet^-1, respectively. CONCLUSION The established method is accurate, simple and rapid, and can be utilized for determination of acarbose in acarbose tablets.     

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??OBJECTIVE To rapidly identify the related substances in doxycycline hyclate tablets and investigate the possible degradation pathways of doxycycline hyclate solution by 2D-LC-IT-TOF/MS. METHODS Firstly,the chromatography was carried out using the 1stD InertSustain^TM C₁₈(4.6 mm??150 mm,5 ??m) column and a mobile phase containing a mixture of buffer solution(0.25 mol??L^-1 ammonium acetate solution-0.1 mol??L^-1 ethyldiaminetetraacetic acid disodium: triethyiamine=100??10??1)-acetonitrile(85??15). Solution pH was adjusted to 8.8 with glacial acetic acid. Then a InertSustain^TM C₁₈(2.1 mm??50 mm,2 ??m) column was used with 0.1% formic acid-water as mobile A and 0.1% formic acid-acetonitrile as mobile B in a gradient elution mode in 2ndD. Electrospray ionization (ESI) source was tested in both positive and negative ion modes. Nebulized gas flow was 1.5 L?? min^-1. Dry gas flow was 10 L?? min^-1. The desolvation tube temperature was kept at 200 ??. Related substances were characterized according to multi-level MS behaviors. The 2D-LC-IT-TOF/MS method was employed to identify the structures of impurities in forced degradation solutions and illuminate the degradation pathways of doxycycline hyclate solution. RESULTS A total of eight related substances were detected in doxycycline hyclate tablets. Four of them had a content of more than 0.1%, and their structures were identified to be 4-epidoxycycline, metacycline, ??-epidoxycycline and 2-acetyl-2-decarbamoyldoxycycline. The solution of doxycycline hyclate easily degraded under alkaline condition and generated an open loop compound taken off the keto group. The solution was sensitive to heat, and generated 4-epidoxycycline with a little amount of 4-epi-6-epidoxycycline; but the solution was stable under illumination and acidic condition. CONCLUSION The established method is suitable for rapid identification of impurities in doxycycline hyclate, which can be applied as a useful analytical tool for quality control and drug process optimization of doxycycline hyclate.     

HPLC-TQ-MS/MS�ⶨ��ζ���ٽ�����6����Ч�ɷֵ�ѪҩŨ�ȼ���ҩ��ѧ�о�

??OBJECTIVE To establish an HPLC-TQ-MS/MS assay for simultaneous determination of berberine, plantamajoside, saikosaponin a, tetrahydropalmatine, paeoniflorin and amygdalin in rat plasma and investigate the pharmacokinetics of Jiawei Baiteng extracts in rats. METHODS The separation was achieved on an Agilent Poroshell 120 EC-C₁₈ column(3.0 mm??100 mm,2.7 ??m) at 35 ??. The mobile phase was consisted of 0.05% formic acid aqueous solution and acetonitrile containing 0.05% formic acid, eluting with a gradient procedure. Mass spectrometry was performed in the multiple reaction monitoring (MRM) mode, with programmed ionization modes witching and time segment scanning. The ion reactions for quantification were as follows: m/z 335.9??m/z 319.9(berberine, ESI⁺), m/z 639.1??m/z 160.9(plantamajoside, ESI^-),m/z 779.3??m/z 617.4(saikosaponin a, ESI^-),m/z 356.0??m/z 192.0(tetrahydropalmatine,ESI⁺),m/z 449.1??m/z 121.1(paeoniflorin, ESI^-),m/z 456.1??m/z 323.1(amygdalin, ESI^-),m/z 323.9??m/z 126.9(gliclazide, internal standard, ESI⁺) and m/z 321.9??m/z 170.1 (gliclazide, internal standard, ESI^-). Blood samples were collected in heparinized tubes via the retinal venous plexus from each rat after a single oral dose of Jiawei Baiteng extracts(3.1 g??kg^-1). The plasma samples were pretreated by methanol precipitation to remove protein components, and then analyzed by HPLC-TQ-MS/MS. The pharmacokinetic parameters of the bioactive components of Jiawei Baitengex tracts in rats were calculated by DAS (Drug and Statistics for Windows) software(Version 2.0). RESULTS The methodological test showed that the linear concentration ranges of berberine, plantamajoside, saikosaponin a, tetrahydropalmatine, paeoniflorin and amygdalin were 0.59-292.50, 0.68-168.75, 6.05-1 512.50, 0.68-337.50, 6.70-1 675.00 and 5.60-1 400.00 ng??mL^-1, respectively. The limits of quantification (LOQs) of the six analytes were 0.59,0.68,6.05,0.68,6.70 and 5.60 ng??mL^-1, respectively. The HPLC-TQ-MS/MS method was also validated with good precision, recovery and stability, which conformed to the analytical standards of biological samples. CONCLUSION The established method is proved to be sensitive, simple and reliable, which is suitable for the pharmacokinetic study of Jiawei Baiteng extracts.     

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??OBJECTIVE To establish an HPLC method for determining four kinds of rare saponins, ie, 20(S)-Rg₃,20(R)-Rg₃, Rk₁, and Rg₅in black ginseng.METHODS COSMOSIL C₁₈-PAQ (4.6 mm×250 mm, 5 μm) column was used and temperature was maintained at 30 ??. Gradient elution was conducted using mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL·min^-1.The detection wavelength was set at 203 nm. The injected sample volume was 10 μL.RESULTS Good resolution was achieved for the four rare saponins in the ranges of 0.051-0.256 (r=0.999 9),0.009-0.280(r=0.999 7),0.051-0.303(r=0.999 9) and 0.093-0.279 mg·mL^-1(r=0.999 9) for 20(S)-Rg₃, 20(R)-Rg₃, Rk₁, and Rg₅, respectively. The corresponding average recovery rates were 103.9%, 99.2%, 97.0%, and 100.6%, and the standard deviations were 0.71%, 0.73%, 1.97%, and 0.57%, respectively.CONCLUSION The method is accurate, simple, reliable and reproducible for the determination of saponins in black ginseng. The determination result can be used as a reference for the rational medication, quality control, and further study of black ginseng.     

��-���ݶ���TxID���ǻ���ͬ��Ȧ�3��4��������������������о�

??OBJECTIVE To interrogate differential sensitivity of ??-conotoxin TxID on stoichiometry of ??3??4 nicotinic acetylcholine receptors(nAChRs). METHODS Oocytes of Xenopus laevis were used to express rat ??3??4 nAChRs with different stoichiometries by altering ??3:??4 RNA injection ratios of 1:1, 1:10 or 10:1. Sensitivity of ??-conotoxin TxID on these different stoichiometry receptors were evaluated and compared. RESULTS The three stoichiometry receptors of ??3??4 nAChRs were expressed in oocytes successfully. ??-Conotoxin TxID showed differential sensitivity on ??3??4 nAChR stoichiometries. Inhibition of 1:10 injection ratio by TxID was similar with regular 1:1 ??3??4 nAChRs within 2-fold difference. While potency of 10:1 injection ratio by TxID decreased 5-fold significantly comparing with 1:1 ??3??4 nAChRs. CONCLUSION ??-Conotoxin TxID exhibits distinct sensitivity on different stoichiometry of ??3??4 nAChRs, which could reflecting different stoichiometries of ?? and ?? subunits. The RESULTS would be helpful for elucidation of structure and physiology function of ??3??4 nAChRs.     

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??OBJECTIVE To study the generality of the structure-activity relationships(SARs)of 3-deoxylation and 6-deoxylation of the sugar moiety in SGLT2 inhibitors. METHODS Based on the earlier study, 3-deoxycanagliflozin(compound 5), 6-deoxyipragliflozin(compound 6), 6-deoxyempagliflozin(compound 7)and 3-deoxyempagliflozin(compound 8)were synthesized and evaluated by in vitro hSGLT2 and hSGLT1 inhibitory assay. RESULTS The deoxylated SGLT2 inhibitors were synthesized from their corresponding aryl halides 9a-9c and 3-/6-deoxylated perbenzylated gluconolactones 11a-11b.In vitro hSGLT2 and hSGLT1 inhibitory assay showed that compounds 5 and 8 almost lost SGLT2 inhibitory activity, while compounds 6 and 7 exhibited similar activities to their corresponding parent compounds. CONCLUSION It seems a general rule that 6-deoxylation of the sugar moiety in SGLT2 inhibitors has no effect on the SGLT2 inhibitory activity, whereas the effect of 3-deoxylation on SGLT2 inhibitory activity depends on the structures of the specific SGLT2 inhibitors, which does not show a universal rule.
     

��-���ݶ���TxIB�칹��Դ���������6/��3��2��3��������������׿������о�

??OBJECTIVE To investigate antagonistic activities of three isomers of ??-conotoxin TxIB on rat and human ??6/??3??2??3 nicotinic acetylcholine receptors (nAChRs). METHODS Three disulfide bond isomers were synthesized using Fmoc chemistry, which were identified by ultra performance liquid chromatography (UPLC)and confirmed by MALDI-TOF mass spectrometry. Rat and human ??6/??3??2??3 nAChRs were expressed in oocytes of Xenopus laevis, which were used to test the antagonistic abilities of the 3 isomers. RESULTS The three isomers of ??-conotoxin TxIB were synthesized successfully.The retention time of each isomer of ??-conotoxin TxIB was different each other significantly. The observed molecular masses of three isomers were the same, which were consistent with their theoretical molecular mass.Their hydrophilicity orders were globular > ribbon> bead. Both rat and human ??6/??3??2??3 nAChRs were expressed in oocytes well. Inhibition of three isomers of ??-conotoxin TxIB on rat and human ??6/??3??2??3 nAChRs were evaluated respectively. Among the three isomers of TxIB, the activity of the globular isomer was the most potent one, which had almost same activity at rat and human ??6/??3??2??3 nAChRs with corresponding IC₅₀ of 28.2 and 32.0 nmol??L^-1respectively. However, the other two isomers, ribbon and bead isomers displayed little antagonistic effect on both rat and human ??6/??3??2??3 nAChRs only with an IC₅₀ of ??10 ??mol??L^-1. CONCLUSION The synthesized globular isomer of ??-conotoxin TxIB in this work has a high selectivity and potent antagonistic activity on rat and human ??6/??3??2??3 nAChRs, which would be helpful for its new drug development.     

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??OBJECTIVE To study the chemical constituents of Swertia binchuanensis. METHODS The constituents were isolated and purified by column chromatography of silica gel. Their structures were identified on the basis of spectral analysis and chemical properties. RESULTS Five compounds are isolated and identified as 7-O-????-L-rhamnopyranosyl-(1??2)-??-D-xylopyranosyl??-1,8-dihydroxy-3-methoxyxanthone(1), 3-O-??-D-glucopyranosyl-1,8-dihydroxyl-5-methoxyxanthone(2), 7-O-??-D- glucopyranosyl-1,8-dihydroxyl-3-methoxyxanthone(3), amarogentin(4), and amaroswerin(5). CONCLUSION All of the compounds were isolated from S.binchuanensis for the first time.     

��ЧҺ��ɫ�׷�ͬʱ�ⶨ�˲ν�Ƣ�����˲�����Rg1��Re��Rb1������������A��B�ĺ���

??OBJECTIVE To establish an HPLC method for simultaneous determination of ginsenoside Rg₁, Re, Rb₁ and jujuboside A, B in Renshen Jianpi Pellets (RSJPW). METHODS The determination was conducted on an Eclipse XDB C₁₈ column (4.6 mm??250 mm, 5 ??m) with the mobile phase of acetonitrile (A)-water (B) gradiently eluted at the following flow rates:0-90 min, 1.0 mL??min^-1; 90-130 min, 1.0-0.5 mL??min^-1; 130-140 min, 0.5 mL??min^-1. And the column temperature was maintained at 35 ??; the detection wavelength was set at 203 nm. RESULTS The calibration curves of ginsenoside Rg₁, Re, Rb₁ and jujuboside A, B were in good linearity over 0.32-2.24 ??g (r=0.998 2), 0.16-1.12 ??g (r=0.995 3), 0.32-2.24 ??g (r=0.999 6), 0.16-1.12 ??g (r=0.991 5), 0.08-0.56 ??g (r=0.999 6). The corresponding average recovery rates were 97.18%, 97.62%, 98.79%, 98.48%, 94.51%; the standard deviations were 1.10%, 0.98%, 0.34%, 1.09%, 1.88%, respectively. CONCLUSION The established HPLC method is accurate, reliable and reproducible, which can be used as a reference for the quality control of RSJPW.     

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??Endogenous neurotoxin 1-methyl-6,7-dihydroxy-1,2,3,4 -tetrahydroisoquinoline (salsolinol,Sals), an endogenous dopamine metabolite, were shown to be toxic to dopaminergic neurons in vitro as well as in vivo, and was known to be involved in the pathogenesis of Parkinson??s disease (PD). Sals is a more realistic model for selective toxicity to nigral dopaminergic neurons, and mimic the natural course of PD that develops slowly, allowing the brain to adapt to progressive damage. Sals lead to neurotoxicity in dopaminergic cells through induction of oxidative stress and apoptotic dopaminergic cell death,which made it as an important tool drugs to study the pathogenesis of PD.     

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??OBJECTIVE To study the chemical constituents of Patrinia villosa (Thunb.) Juss. METHODS The compounds were isolated by a combination of various chromatographic techniques including column chromatography over macroporous resin, Sephadex LH-20, and reversed-phase HPLC. Their structures were elucidated by physiochemical property and spectral analysis. RESULTS Eleven compounds were isolated and identified as(7R,8S)-3,3??,5-trimethoxy-4??,7-epoxy-8,5??-neolignan-4,9,9??-triol-9-O-??-D-glucopyranoside(1), massonianoside D(2),(7R,8S)-dihydroxydehydrodiconiferyl alcohol-4-O-??-D-glucopyranoside(3),(7S,8R)-dihydroxydehydrodiconiferyl alcohol-4-O-??-D-glucopyranoside(4), 7R,8S-glochidioboside(5), lariciresinol-4-O-??-D-glucopyranoside(6), lariciresinol-9-O-??-D-glucopyranoside(7), lariciresinol-4??-O-??-D-glucopyranoside(8), tortoside B(9), tanegool(10), and tanegool-7??-methyl ether(11). CONCLUSION All compounds are isolated from Patrinia genus for the first time.     

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??OBJECTIVE To develop on-line high performance liquid chromatography-biochemical detection (HPLC-BCD) methods to screen effective components in Ginkgo biloba extract for treatment of Alzheimer's disease. METHODS Radical scavengers and AchE inhibitors in G.biloba leave extract were screened by HPLC-UV-DPPH/ABTS and HPLC-UV-AchE methods, respectively. The stability and repeatability of the on-line HPLC-UV-AchE method were investigated using galanthamine as a positive reference. The main active ingredients in the extract were identified by ultra high performance liquid chromatography linear ion trap/orbitrap high resolution mass spectrometry (UPLC-LTQ/orbitrap MS) method. RESULTS Fourteen antioxidants in G.biloba extract were detected by the HPLC-UV-DPPH/ABTS method, while three AchE inhibitors were found by the HPLC-UV-AchE method. The three AchE inhibitors were tentatively identified by UPLC-LTQ/orbitrap MS as 4-O-beta-glucopyranosyl-cis-coumaric acid?? ginkgolide A and 3-O-[2-O-(β-D-Glucosyl)-α-L-rhamnosyl]quercetin, respectively. CONCLUSION The proposed on-line HPLC-BCD method shows high sensitivity, good repeatability and stability. It can be used to screen antioxidants and AchE inhibitors in G. biloba extract. This will provide an effective, quick and convenient analysis tool to quickly find bioactive ingredients of traditional Chinese medicines for treating related diseases.     

��-����������ȫ��ʽά������ת�˰�Ѫ��ϸ��K562/ADM��ҩ�Ե��о�

??OBJECTIVE To investigate the effects of interferon-??(IFN-??) and all-trans retinoic acid(ATRA) on multidrug resistance reversal effect and mechanism of human leukemia K562/ADM cells. METHODS The cytotoxicity and reversal times of IFN-?? and ATRA were detected by CCK-8 method. Apoptosis rate and cell cycle were detected by flow cytometry. PI3K, Akt and Bad mRNA were detected by RT-PCR method. Western blot method was used to detect the expression of PI3K, AKt, P-AKt and Bad protein.RESULTS The drug resistance of K562/ADM cells to adriamycin(ADM) was 54 times. ADM, respectively, with IFN-??, ATRA or combined application, the drug resistance of K562/ADM cells to ADM was 1.24, 2.34 and 8.14, respectively. The apoptosis rate of K562/ADM cells was significantly increased by using ADM 4 mg??L^-1alone or in combination with IFN-?? 2.5??10⁶ U??L^-1, ATRA 7.5 ??mol??L^-1, and the cell cycle was blocked in G₀/G₁ phase. PI3K mRNA and protein expression were significantly lowered, Akt mRNA and protein has no obvious change, Bad mRNA and protein expression are raised, phosphorylated Akt protein expression decreased, the expression is more obvious when the two drug combination. CONCLUSION IFN-?? and ATRA can reverse the multidrug resistance of K562/ADM cells, its mechanism may be the inhibition of the PI3K/Akt pathway.