A Rapid Method to Screen for Multi Drug Resistance (MDR) Trait in Clinical Isolates of Acinetobacter baumannii-Acyl Homoserine Lactone (AHL) Screening in CRAB and CSAB Isolates

Aims: Our proposal aimed to evaluate Acyl Homoserine Lactones (AHL) as a functional marker for Multi drug resistant (MDR) potential in clinical isolates of Acinetobacter baumannii. We investigated the AHL production potential of clinical isolates using a biosensor assay directly on a commonly used agar media. Place and Duration of Study: Department of Molecular Diagnostics and Biomarkers, Gleneagles Global Hospitals, Lakdikapul, Hyderabad-500004. Methodology: Antimicrobial drug sensitivity testing (AST) was performed on 72 clinical isolates of A. baumannii against two front-line antibiotics, Imipenem (10µg) and Meropenem (10µg), by Kirby-Bauer disk diffusion method. Production of long chain Acyl Homoserine lactone (AHLs) in the clinical isolates of A. baumannii was tested by cross streaking with the biosensor Chromobacterium violaceum mutant strain CV026 and Agrobacterium tumefaciens (NTL4pZLR4) by agar plate diffusion assay. Screening and identification of the quorum sensing mediator gene abaI was done by PCR to confirm its presence in all the 72 clinical isolates. Results: Out of the 72 clinical isolates, 58 were Carbapenem resistant Acinetobacter baumannii (CRAB) and 14 were Carbapenem sensitive Acinetobacter baumannii (CSAB) for AST by agar disc diffusion method. None of our isolates produced short chain AHLs whereas all the isolates could produce varying amounts of long chain AHLs. Genotypic confirmation of AHL gene was obtained by abaI gene PCR. Conclusion: Carbapenems are the front-line antibiotics used to treat gram negative bacterial infections in emergencies and in the critical care units of hospitals. Clinical isolates A. baumannii has innate resistance to several antibiotics due to various mechanisms, biofilms forming the first line of defense against antibiotics for the bacterium. Our study used AST to carbapenem as the leading marker for MDR, assuming the innate resistance of A. baumannii to other beta lactam antibiotics. Our study brought out certain important observations namely: a) All clinical isolates of A. baumannii produced Quorum Sensing signal molecules, the AHLs b) the clinical isolates of A. baumannii did not produce any short chain AHLs b) All the clinical isolates of A. baumannii produced long chain AHLs c) AHL production is not specific to carbapenem drug resistance because even CSAB isolates produced AHL d) AHL production is inherent to all clinical isolates of A. baumannii and it apparently indicates an underlying biofilm potential and MDR trait in these A. baumannii isolates. e) AHLs could be a universal marker for revealing MDR trait and biofilm potential in clinical microbiology AST profiling protocols.

Establishment and application for simultaneous determination method of atorvastatin and its active/toxic metabolites in rat plasma / 中国药房

OBJECTIVE To establish a method for simultaneous determination of atorvastatin （ATV） and its active metabolites 2-hydroxy atorvastatin acid （2-HAT）， 4-hydroxy atorvastatin acid （4-HAT） and toxic metabolite atorvastatin lactone （ALT） in rat plasma and apply it for pharmacokinetic study. METHODS LC-MS/MS method was adopted for analysis. The one-step precipitation method was used for processing plasma samples （plasma samples were pretreated by acidification to adjust pH value so as to prevent inversion of configuration）， gradient elution was used to analyze the samples， and the analysis time was 5 min. Electrospray positive ionization was adopted， and positive ion scanning was performed in multi-reaction monitoring. The m/z of quantified ion pairs of ATV and its metabolites such as 2-HAT， 4-HAT and ATL， and internal standard pitavastatin were 559.3→ 440.2， 575.2→440.3， 575.0→440.2， 540.9→448.2 and 422.2→290.0， respectively. After conducting a comprehensive methodological investigation of the analytical method， the concentrations of ATV and its metabolites 2-HAT， 4-HAT，and ATL were determined， and the pharmacokinetic parameters of ATV and its metabolites were calculated using the non- compartment model of WinNonlin 6.1. RESULTS The results of methodological validation showed that endogenous substances in blank plasma did not interfere with the determination of the components to be tested， and the standard curve had a good linear relationship； the lower limits of quantification for ATV， 2-HAT， 4-HAT and ATL were 0.5， 0.5， 0.25 and 0.063 nmol/L， respectively. The precision， accuracy， recovery， matrix effect and stability investigation were all in line with the requirements of biological analysis. Pharmacokinetic analysis showed that after intragastric administration in rats， ATV calcium metabolized rapidly， and was mainly exposed to blood circulation in the form of ATV and 2-HAT， with the lowest concentration of lactone-type metabolites. CONCLUSIONS The established method is precise， rapid and accurate for plasma concentration analysis of ATV and its active/toxic metabolites. The application of the method could help to fully elucidate the pharmacokinetic characteristics of atorvastatin calcium in rats.

Role of calcium-independent phospholipase A2 in contraction of intrarenal artery smooth muscle / 中国药理学通报

Aim To investigate the role of calcium-independent phospholipase A2(iPLA2)in calcium regu-lation of intrarenal artery smooth muscle contraction.Methods The method of measuring the tension of isolated arterioles was used to explore the effect of bromoenol lactone(BEL), a specific inhibitor of iPLA2, on the tension of the intrarenal arteries in mice induced by different calcium channels, and the laser confocal calcium measurement technology was used to investigate the effect of BEL on the intracellular calcium influx mediated by arachidonic acid-mediated calcium channels.Results The intrarenal artery concentration dependent contractile response induced by the vasoconstrictors phenylephrine and 5-hydroxy tryptamine was inhibited by BEL(P<0.01).The contraction curve induced by CaCl2 was also inhibited by BEL(P<0.05).In the calcium-free K-H solution incubated with nifedipine, the intrarenal artery vasoconstriction caused by the release of sarcoplasmic reticulum calcium and the calcium influx of the SOC channel induced by CaCl2 was inhibited by BEL(P<0.05).BEL significantly inhibited the external calcium influx mediated by the ARC channel of human aortic smooth muscle cell lines incubated with nifedipine(P<0.01).Conclusions iPLA2 mediates the contractile response of intrarenal arteries by regulating the functions of L-type calcium channels, sarcoplasmic reticulum calcium release, SOC channels and ARC channels.

A new seco-prezizaane-type sesquiterpene lactone from stems and branches of Illicium ternstroemioides / 中国中药杂志

Three seco-prezizaane-type sesquiterpene lactones, one phenylpropanoid, and two lignans were isolated from the 95% ethanol extract of stems and branches of Illicium ternstroemioides with silica gel column chromatography, ODS column chromatography, and preparative HPLC. Based on the spectral data, they were identified as burmanicumolide D(1), veranisatin A(2), veranisatin B(3), dihydroconiferylalcohol(4), pinoresinol(5),(-)-matairesinol(6), respectively. Among them, compound 1 was a new seco-prezizaane-type sesquiterpene lactone, and 2-6 were obtained from this plant for the first time. None of these compounds display antiviral or cytotoxic activities.

Pharmacokinetic study on five components in phthalide target areas of Chaxiong and its β-CD inclusion compounds based on UPLC-MS/MS / 中国中药杂志

This study aims to establish a method for the determination of the concentration of five main components of phthalide target areas of Chaxiong(CPTA) and its inclusion of β-CD in the plasma of rats, and determine the pharmacokinetic parameters, absolute bioavailability and relative bioavailability of CPTA/β-CD inclusion compound in vivo. The plasma concentrations of senkyunolide A, N-butylphthalide, new osthol lactone, Z-ligustilide and butenyl phthalide were determined with UPLC-MS/MS. The content determination was conducted at the chromatographic conditions as follows: Shim-pack GIST C_(18)-AQ HP column(2.1 mm×100 mm, 3 μm), mobile phase of 0.1% formic acid solution(A)-acetonitrile(B), gradient elution, flow rate of 0.3 mL·min~(-1), column temperature of 35 ℃ and injection volume of 2 μL. The mass spectra were obtained with electrospray ion source(ESI), positive ion mode and multi reaction monitoring. CPTA/β-CD inclusion compound was prepared by grinding method, DAS 2.0 software was used to model the data, and the absolute bioavailability of CPTA and relative bioavailability of inclusion compound were calculated. Finally, the methods for the determination of five components of senkyunolide A, N-butylphthalide, new osthol lactone, Z-ligustilide and butenyl phthalide in CPTA, were successfully established. The linear relationship among the five components was good within their respective ranges, r>0.99. The absolute bioavailability of the five components in rats was 22.30%, 16.32%, 21.90%, 10.16% and 12.43%, respectively. After CPTA/β-CD inclusion was prepared, the relative bioavailability of the five components was 138.69%, 198.39%, 218.01%, 224.54% and 363.55%, respectively, significantly improved. This method is rapid, accurate and sensitive, so it is suitable for the pharmacokinetic study of extracts in traditional Chinese medicine and their preparations.

Effects of angelica lactone combined with Xiaoke pill on regulation of vascular endothelial growth factor signal pathway and on renal protection in diabetic nephropathy rats / 解剖学报

Objective To investigate the effects of angelica lactone combined with Xiaoke pill on blood glucose regulation, renal protection and vascular endothelial growth factor signal pathway in diabetic nephropathy rats. Methods Totally 75 rats were randomly divided into control group, model group, Xiaoke pill group, angelica lactone group and angelica lactone combined with Xiaoke pill group, with 15 rats in each group. Except for the control group, the rest of the rats established diabetic nephropathy model. Rats in Xiaoke pill group, angelica lactone group and angelica lactone combined with Xiaoke pill group were given Xiaoke pill (0.8 g/kg), angelica lactone (20 mg/kg), Xiaoke pill (0.8 g/kg) combined with angelica lactone (20 mg/kg), respectively, once for 8 weeks. The levels of 24-hour urinary protein, fasting and postprandial blood glucose, glycosylated serum albumin and glycosylated hemoglobin, fasting insulin and insulin sensitivity index, serum creatinine (SCr) and urea nitrogen (BUN) were measured. The levels of insulin (INS) and glucose-6-phosphatase (G6Pase) mRNA in islet tissue were measured by Real-time PCR. Renal histopathological changes were detected by HE staining. The levels of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) in rat kidney were detected by Western blotting and immunohistochemistry. Results Compared with the model group, 24-hour urinary protein, fasting and postprandial blood glucose, glycosylated serum albumin and glycosylated hemoglobin, fasting insulin, SCr and BUN, VEGF and VEGFR2 protein levels and relative integral absorbance of kidney tissue in angelica lactone combined with Xiaoke pill group were significantly lower than those in model group(P<0.05). The insulin sensitivity index, the levels of INS and G6Pase mRNA in islet tissue were significantly increased (P < 0.05), and the renal histopathology of diabetic nephropathy rats was significantly improved. Conclusion Angelica lactone combined with Xiaoke pill can reduce blood glucose level, increase insulin sensitivity index, and its mechanism may be related to the regulation of VEGF signal pathway.

Simultaneous determination of six components in Mongolia medicine Gurigumu-13 Pills by QAMS / 中草药

Objective: To establish a quantitative analysis of multi-components by single marker (QAMS) method for the simultaneous determination of six components of gallic acid, hydroxysafflor yellow A, geniposide, ellagic acid, costunolide and dehydrocostus lactone in Gurigumu-13 Pill, which is proved to be a scientific and feasible method in the quality analysis in Gurigumu-13 Pill. Methods: The relative factor (fs/i) of gallic acid, ellagic acid, hydroxysafflor yellow A, costunolide and dehydrocostus lactone were established by HPLC method with geniposide as internal standard, which were used to calculate the content of five constituents in the samples of Gurigumu-13 Pill. Meanwhile, external standard method (ESM) was used to calculate the content of six constituents. The difference between QAMS and ESM was analyzed to evaluate the accuracy of QAMS. Results: The fs/i of gallic acid, hydroxysafflor yellow A, ellagic acid, costunolide and dehydrocostus lactone were 0.481 0, 0.906 4, 0.170 9, 0.971 2 and 1.261 5, respectively. The content determination results of six batches of Gurigumu-13 Pill were calculated by the method of QAMS and ESM, with no significant difference in RSD < 2.0%. Conclusion: The fs/i established in the QAMS method with geniposide as the internal reference substance is accurate and simple. The QAMS method can be used for the multi-index quality evaluation of Gurigumu-13 Pill.

A new lactone from Euscaphis konishii / 中草药

Objective: To study the chemical constituents of Euscaphis konishii. Methods: The chemical constituents from 95% EtOH extract of the stems of E. konishii were isolated by repeated chromatograph with silica gel, Sephadex LH-20 and semi-preparative RP-HPLC. The structure of the separated chemical components was identified by modern spectroscopy identification. Results: Eight compounds were isolated from the ethyl acetate fraction of 95% EtOH extract of E. konishii, and identified as 4 (R)-(4'-hydroxybenzoyl) dihydrofuran-2(3H)-one (1), 6-hydroxymellein (2), euscapholide (3), dehydrovomifoliol (4), ethyl gallate (5), tetraketide (6), p-hydroxybenzoic acid (7) and nicotinic acid (8). Conclusion: Except for compounds 3 and 6, they are all isolated for the first time in this genus, and all compounds are isolated for the first time in this plant. Among them, compound 1 is a new compound named konieuscaphide.

Brusatol: A potential anti-tumor quassinoid from Brucea javanica / 中草药·英文版

Brusatol, a triterpene lactone compound mainly from Brucea javanica, sensitizes a broad spectrum of cancer cells. It is known as a specific inhibitor of nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway. In this review, we provide a comprehensive overview on the antitumor effect and molecular mechanisms of brusatol in vitro and in vivo. This review also covers pharmacokinetics studies, modification of dosages forms of brusatol. Increasing evidences have validated the value of brusatol as a chemotherapeutic agent in cancers, which may contribute to drug development and clinical application.

Effect of Mixed Inoculation of Arbuscular Mycorrhizal on Growth and Chemical Composition of Aucklandia lappa Seedlings / 中国实验方剂学杂志

Objective:To explore the synergistic effect of arbuscular mycorrhizal（AM） fungi mixed inoculation on the growth，physiological and biochemical characteristics，root biomass and terpenoid component accumulation of Aucklandia lappa seedlings，so as to provide a reference for the combination and application of the dominant complementary effect mycorrhizal fungi. Method:The effect of different AM fungi combined with inoculation on the root mycorrhizal infection rate，plant growth，physiological and biochemical characteristics，root biomass，costunolide and dehydrocostus lactone of A. lappa seedlings were determined by pot inoculation at room temperature. Result:It was found that AM fungi could form good mycorrhizal symbiosis with the roots of A. lappa.The formation of mycorrhizal symbiosis system could increase the chlorophyll content of A. lappa leaves，increase the activities of catalase（CAT），peroxidase （POD），superoxide dismutase （SOD），reduce the content of malondialdehyde（MDA），and promote photosynthesis of A. lappa. Compared with CK group，AM fungus treatment could significantly promote the accumulation of costunolide and dehydrocostus lactone，and the accumulation of its metabolites，costunolide and dehydrocostus lactone，into roots during the symbiotic cultivation of A. lappa seedlings，indirectly improving the quality of medicines and yield of alantolactone. Conclusion:Inoculation of AM fungi can improve the root mycorrhizal viability，increase the absorption of nutrients and promote the growth of woody incense.The mixed inoculation treatment of S2，S4 and S5 had the best mycorrhizal effect in artificial cultivation，and the growth and medicinal quality of A. lappa were the best，which provided technical support for the application and popularization of A. lappa mycorrhizal biotechnology.

Antiproliferative and photoprotective activities of the extracts and compounds from Calea fruticosa

In this paper, we complement our previous study on the antiproliferative activity of Calea fruticosa (Asteraceae) by isolating the compounds apigenin-4',7-dimethyl ether (1), budlein A (2), quercetin (3), and cichoriin (4) from the plant's aerial parts. The antiproliferative activity of these compounds was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method against human tumor cell lines. Compound 3 displayed moderate antiproliferative activity in three cell lines (HCT-116, PC-3, and SF-295, with cell growth inhibition values of 72.97, 74.55, and 68.94%) and high antiproliferative activity (90.86%) in the HL-60 cell line. The in vitro sun protection factor (SPF) of the extracts and compound 4, with and without sunscreen, was determined by a spectrophotometric method. The ethanol extract exhibited the highest SPF (9.67) at a concentration of 0.100 mg/mL, while compound 4, isolated from this extract, showed a SPF of 13.79 at the same concentration. A relative increased efficacy of SPF was observed for the extracts and compound 4 when sunscreen was also used. Compound 4 has not been reported previously from any species within the genus Calea. Compounds 1-4 were obtained from this species for the first time.

Anticancer activity of britannin through the downregulation of cyclin D1 and CDK4 in human breast cancer cells

Aim of the Study: Both apoptotic induction and cell cycle blockade in cancer cells are effective strategies to eliminate cancer cells. Many conventional cancer drugs that induce apoptosis and inhibit cell cycle progression have been reported as potential therapeutics for various types of cancer. Britannin is a natural sesquiterpene lactone that its profound anticancer properties were revealed in our previous study. In this study, we evaluated the effects of britannin on the cell cycle distribution and also cell cycle-related proteins. Materials and Methods: Analysis of cell cycle distribution was carried out using flow cytometer. The effects of britannin on cyclin D1 and CDK4 expression were evaluated using the Western blot. Results: The obtained results show that britannin at the low concentrations induces cell growth inhibition mainly through G1-phase arrest while it seems that apoptosis contributes to cell growth inhibitory effect of high doses of britannin. Reduction of cyclin D1 and CDK4 protein levels were also observed after treating cancer cells with britannin. Conclusion: The obtained results reveal that britannin can inhibit MCF-7 and MDA-MB-468 breast cancer cells proliferation through arresting cell cycle progression through cyclin D1/CDK4-mediated pathway

Dose dependant anti-inflammatory effects of Dehydrocostus Lactone / Монголын Анагаах Ухаан

Introduction@#After central nervous system injury, microglia cells are activated to initiate inflammatory responses and release cytokines that beneficially or detrimentally affect surrounding cells. Lipopolysaccharide stimulates microglia cells and produce pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α. A dehydrocostus lactone (DDL) which is contained in medicinal plant, Saussurea lappa, is considered to have various health benefits in neurodegenerative diseases of central nervous system. </br> In this study, we aimed to investigate the anti-inflammatory effects of Dehydrocostus Lactone following lipopolysaccharide stimulation of microglial cells in vitro.@*Materials and Method@#The anti-inflammatory effects of dehydrocostus lactone were studied using lipopolysaccharide (LPS) stimulated murine microglia (BV2). BV2 were cultered in DMEM then three different doses (4µM, 8µM and 12µM) of DDL were added in the medium for 30 minutes respectively. Then BV2 were treated with 1 ng/ ml LPS for 24 hours to stimulate. The level of IL-1β, IL-6 and TNF-α were measured in 100µl of culturemedium supernatant by ELISA. Three different doses of DDL anti-inflammation groups (BV2+DDL+LPS), LPS-activated group (BV2+LPS) and control group (only BV2) were analysed. @*Results@#LPS-treated BV2 cells had increased IL-1β, IL-6 and TNF-α compared with those without LPS treatment. Pretreatment with dehydrocostus lactone prior to LPS treatment significantly decreased levels of IL-1β and TNF-α in a dose-dependent manner compared with LPS-treated BV2 cells and 4µM was the most effective anti-inflammatory dose of dehydrocostus lactone. As for IL-6, 12µM dehydrocostus lactone was the most effective anti-inflammatory dose, although all doses significantly decreased the level of IL-6, in a dose-dependent manner. @*Conclusion@#These results show that DDL decrease inflammation related IL-1β, IL-6 and TNF-α in a dose-dependent manner in microglia cells.

Preparation and characterization of PEGylated long-circulating liposomes encapsulating costunolide and dehydrocostus lactone / 中草药

Objective: To prepare pegylated long-circulating liposomes co-encapsulated by costunolide (Cos) and dehydrocostus lactone (DL), optimize the formulation and process, and evaluate the quality. Methods: The pegylated long-circulating liposomes co-encapsulated by Cos and DL were prepared by film hydration method. Single factor test and Box-Behnken response surface methodology were used to optimize the preparation process with encapsulation efficiency of Cos and DL as the index. The particle size, surface potential, encapsulation efficiency and in vitro release of the liposomes were evaluated. Results: The optimal preparation conditions were as follows: drug-to-lipid ratio was 0.14, ratio of cholesterol to phospholipid was 0.05, mPEG-2000-DSPE addition amount was 6%, hydration time was 30 min, and probe ultrasonic time was 4 min. The obtained liposome was round and uniform in distribution, with an average particle size of (104.8 ± 2.48) nm, a polydispersity index (PDI) of (0.245 ± 0.031), and a Zeta potential of (-9.7 ± 0.23) mV, the encapsulation efficiency of Cos and DL were (91.9 ± 2.6)% and (94.41 ± 1.23)%, respectively. Conclusion: The PEGylated long-circulating liposome prepared by the process and prescription optimization has good appearance and high encapsulation efficiency, which can meet the application requirements.

A new lactone from Alternaria sp. IS275, an endophytic fungus of Ligusticum chuanxiong / 中草药

Objective: To study the chemical constituents of metabolites of Alternaria sp. IS275, an endophytic fungus isolated from Ligusticum chuanxiong. Methods: The extraction fraction of ethyl acetate from endophytic fungi fermentation broth of L. chuanxiong were isolated and purified by silica gel, Sephadex LH-20, reversed phase medium pressure chromatography, preparative TLC, and semi-preparative RP-HPLC. The structure of the separated chemical constituent was elucidated by modern spectroscopy technology. Results: A new lactone compound (+)-4,11-dihydroxy-2-dodecene-5-lactone was isolated from the ethyl acetate fraction of the metabolite of Alternaria sp. IS275, an endophytic fungus of L. chuanxiong. Conclusion: Compound 1 is a new lactone compound, named ligualterlide A.

Simultaneous Determination of 6 Components in Libiling Tablets by RP-HPLC / 中国药房

OBJECTIVE： To establish a method for simultaneous determination of matrine， oxymatrine， gallic acid， peoniflorin， costunolide and dehydrocostus lactone in Libiling tablets. METHODS： RP-HPLC method was adopted. The determination was performed on Agilent ZORBAX SB-C18 column with mobile phase consisted of methanol-0.1％ phosphoric acid （gradient elution） at the flow rate of 1.0 mL/min. The detection wavelengths were 210 nm （matrine， oxymatrine） and 225 nm （gallic acid， peoniflorin， costunolide， dehydrocostus lactone）. The column temperature was set at 30 ℃， and sample size was 5 μL. RESULTS： The linear ranges of matrine， oxymatrine， gallic acid， peoniflorin， costunolide， dehydrocostus lactone were 0.053-5.28 mg/mL（r＝0.999 8）， 0.125-12.54 mg/mL（r＝0.999 9）， 0.013-1.33 mg/mL（r＝0.999 8）， 0.169-16.94   mg/mL（r＝0.999 9）， 0.048-4.77 mg/mL（r＝0.999 8）， 0.072-7.16 mg/mL （r＝0.999 9）. The limits of quantitation were 4.08×10－4， 4.48×10－4， 3.12×10－4， 2.10×10－4， 1.36×10－4， 1.84×10－4 mg/mL， respectively. The limits of detection were 1.24×10－4， 1.50×10－4， 1.02×10－4， 6.20×10－5， 4.20×10－5， 6.40×10－5 mg/mL， respectively. RSDs of precision， stability and reproducibility tests were all lower than 2％ （n＝6）. The recoveries were 98.03％-101.43％ （RSD＝1.25％， n＝6）， 97.73％-102.26％ （RSD＝1.96％， n＝6）， 97.18％-101.41％ （RSD＝1.98％，n＝6）， 97.45％-102.11％ （RSD＝1.88％，n＝6）， 96.85％-101.07％ （RSD＝1.75％， n＝6）， 97.12％-102.64％ （RSD＝1.82％，n＝6）， respectively. CONCLUSIONS： Established method is simple， stable and rapid， and can be used for simultaneous determination of 6 components in Libiling tablets.

Separation and Purification of Costunolide and Dehydrocostus Lactone in Aucklandiae Radix by Supercritical Fluid Chromatography / 中国实验方剂学杂志

Objective:To establish a supercritical fluid chromatography(SFC) method for separating and purifying costunolide and dehydrocostus lactone in Aucklandiae Radix. Method:With supercritical carbon dioxide as the mobile phase,the effect of six factors, such as type of chromatographic columns,modifiers and modifiers ratio, flow rate of mobile phase,pressure and temperature, on the separation process of supercritical fluid chromatography were explored. The target components were separated and prepared by semi-preparative supercritical fluid chromatography. High performance liquid chromatography and nuclear magnetic resonance were used to analyze the components and study the thermodynamic regularity of the chromatographic process. Result:C18 column (10 mm×250 mm,5 μm) was adopted, with supercritical fluid dioxide as the mobile phase,the ratio of methanol was 0.13%,the flow rate was 12 mL·min-1,column pressure was 13 MPa,column temperature was 318℃, and detection wavelength was 225 nm. The sample was injected for 20 times,crude extract was 4 mg,and each target component was collected according to the chromatogram. Its purity was determined to be more than 99%by HPLC,and its structure was determined as costunolide and dehydrocostus lactone by NMR. Under this condition,the SFC separation process was normal-phase chromatography. Conclusion:The method can be used to prepare effective components of Aucklandiae Radix with a high purity and low solvent residue.

Monomeric and dimeric sesquiterpene lactones from Artemisia heptapotamica / 中国天然药物

One new dimeric (1) and two monomeric sesquiterpene lactones (5 and 13), together with 10 known compounds (2-4, 6-12), were isolated from Artemisia heptapotamica collected in Almaty region of Kazakhstan. All compounds were isolated from this plant for the first time. The structures of the new compounds were mainly achieved by extensive analysis of MS, 1D and 2D NMR spectroscopic data, and ECD spectrum as well. The inhibitory activities of all isolates against activation of NF-κB induced by LPS were assessed on a THP1-Dual cell model. Some of them showed strong inhibitory activity with IC values ranging from 2 to 25 μmol·L.

Effect of dehydrocostus lactone on the proliferation of chronic myeloid leukemia K562 cells and its mechanism / 白血病·淋巴瘤

Objective To explore the effect of dehydrocostus lactone on the proliferation of chronic myeloid leukemia cell line K562 and its mechanism.Methods K562 cells in logarithmic growth phase were treated with different concentrations of dehydrocostus lactone.Cell morphology was observed by Wright-Giemsa staining.Cell proliferation was detected by CCK-8 method.Cell cycle,apoptosis and the expressions of cell surface differentiation antigen CD14 and CD1 1b were detected by flow cytometry.JAK-STAT pathway and the expressions of apoptosis and cell cycle related proteins were detected by Western blot.Results Compared with the control group,the proliferation of K562 cells could be inhibited after treatment with different concentrations (4.0,6.0,8.0,10.0 and 12.0 μmol/L) of dehydrocostus lactone for 24 h,and the difference was statistically significant (F =109.510,P ＜ 0.05).After treatment with 5.0 and 10.0 μmol/L dehydrocostus lactone for 24 h,the apoptosis rates of K562 cells were (16.1±3.8)％ and (29.6±4.3)％,which were higher than that of the control group [(3.1±0.5)％] (F =83.255,P ＜ 0.05).After treatment with 5.0 and 10.0 μmol/L dehydrocostus lactone for 24 h,the proportion of G2/M phase cells in K562 cells were (17.0±3.2)％ and (28.8± 3.9)％,which were higher than that of the control group [(9.1±2.3)％] (F =161.598,P ＜ 0.05);the proportion of S phase cells in K562 cells were (48.1±3.9)％ and (61.0±5.4)％,which were higher than that of the control group [(39.6±3.6)％] (F =192.356,P ＜ 0.05).After treatment with 2.5 and 5.0 μmol/L dehydrocostus lactone for 72 h,the expression rates of CD14 in K562 cells were (28.6±3.9)％ and (41.1±4.4)％,which were higher than that in the control group [(3.1±0.5)％] (F =132.811,P ＜ 0.05);the expression rates of CD11b in K562 cells were (42.4±5.0)％ and (61.2±5.7)％,which were higher than that in the control group [(4.2±1.1)％] (F =179.553,P ＜ 0.05).Dehydrocostus lactone could decrease the expressions of JAK2,STAT5,eyclin E,CDK2,cyclin A,CDC25C,cyclin B1,CDK1 and bcl-2 proteins,and up-regulate the expressions of p21 and bax proteins.Conclusion Dehydrocostus lactone can inhibit the proliferation of chronic myeloid leukemia K562 cells,which may be achieved by cell cycle arrest,induction of apoptosis and differentiation.

Cytotoxic Lactones from the Pericarps of Litsea japonica

From the pericarps of Litsea japonica (Thunb.) Jussieu, eighteen butanolide derivatives (1 – 18) were evaluated for their cytotoxic activity against HeLa, HL-60, and MCF-7 cells. Compounds 1 – 9 with 2-alkylidene-3-hydroxy-4-methylbutanolides structure exhibited cytotoxic activities against cancer-cell lines. Among them, compound 8 (litsenolide D₂) exhibited the most potent cytotoxicity against the tested cell lines, including HeLa, HL-60, and MCF-7, with IC₅₀ values of 17.6 ± 1.3, 4.2 ± 0.2, and 12.8 ± 0.0 µM, respectively. Compound 8 induced apoptosis in a dose-dependent manner. Annexin V/Propidium Iodide (PI) double staining confirmed that 8 effectively induced apoptosis in MCF-7 cells. To the best of our knowledge, we have reported cytotoxic activity of butanolides from L. japonica against these cancer-cell lines for the first time.