Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent.Here,we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme pu-trescine N-methyltransferase(PMT)and the downstream enzyme hyoscyamine6β-hydroxylase(H6H)of scopolamine biosynthesis in transgenic henbane(Hyoscyamus niger)hairy root cultures.Transgenic hairy root lines expressing both pmt and h6h produced significantly higher(P<0.05)levels of scopolamine compared with the wild-type and transgenic lines har-boring a single gene(pmt or h6h).The best line(T 3 )produced411mg/liter scopolamine,which was over nine times more than that in the wild type(43mg/liter)and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H 11 (184mg/liter).To our knowledge,this is the highest scopolamine content achieved through genetic engi-neering of a plant.We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield,which was more efficient than plants harboring only one of the two genes.It seems that the pulling force of the downstream enzyme(the faucet enzyme)H6H plays a more important role in stimulating scopolamine accumulation in H.niger whereas the functioning of the upstream enzyme PMT is increased propor-tionally.This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.

A New Lignan from Boschniakia himalaica

From the ethanol extract of the whole plant of Boschniakia himalaica Hook. f. et. Thoms, a new and two known lignans have been isolated and identified as 7-methoxypinoresino 1, pinoresinol 2, and pinoresinol-O--D-glucopyranoside 3 respectively. Their structures have been established by spectroscopic methods.

Two New C_(21) Steroids from Marsdenia tenacissima

Two new C21 steroids were isolated from the CHCl3 extract of the stem of Marsdenia tenacissima. On the basis of spectroscopic analysis and chemical methods, their structures were elucidated as 17β-tenacigenin B (2) and 3-O-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D- oleandropyranosyl-tenacigenin C (3). The structure of the known aglycon tenacigenin C was revised as 5α, 9α, 17β-pregnane-3β, 8β, 11α, 12β, 14β-pentanol-20-one. Compound 3 is the first reported glycoside of tenacigenin C.

High-density lipoprotein as a potential carrier for delivery of a lipophilic antitumoral drug into hepatoma cells

AIM: To investigate the possibility of recombinant highdensity lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells. METHODS: Recombinant complex of HDL and aclacinomycin (rHDL-ACM) was prepared by cosonication of apoproteins from HDL (Apo HDL) and ACM as well as phosphatidylcholine. Characteristics of the rHDL-ACM were elucidated by electrophoretic mobility, including the size of particles, morphology and entrapment efficiency. Binding activity of rHDL-ACM to human hepatoma cells was determined by competition assay in the presence of excess native HDL. The cytotoxicity of rHDL-ACM was assessed by MTT method. RESULTS: The density range of rHDL-ACM was 1.063-1.210 g/mL, and the same as that of native HDL. The purity of all rHDL-ACM preparations was more than 92%. Encapsulated efficiencies of rHDL-ACM were more than 90%. rHDL-ACM particles were typical sphere model of lipoproteins and heterogeneous in particle size. The average diameter was 31.26±5.62 nm by measure of 110 rHDL-ACM particles in the range of diameter of lipoproteins. rHDL-ACM could bind on SMMC-7721 cells, and such binding could be competed against in the presence of excess native HDL. rHDL-ACM had same binding capacity as native HDL. The cellular uptake of rHDL-ACM by SMMC-7721 hepatoma cells was significantly higher than that of free ACM at the concentration range of 0.5-10 μg/mL (P<0.01). Cytotoxicity of rHDL-ACM to SMMC-7721 cells was significantly higher than that of free ACM at concentration range of less than 5 ug/mL (P<0.01) and IC50 of rHDL-ACM was lower than IC50 of free ACM (1.68 nmol/L vs3 nmol/L). Compared to L02 hepatocytes, a normal liver cell line, the cellular uptake of rHDL-ACM by SMMC-7721 cells was significantly higher (P<0.01) and in a dose-dependent manner at the concentration range of 0.5-10 μg/mL.Cytotoxicity of the rHDL-ACM to SMMC- 7721 cells was significantly higher than that to L02 cells at concentration range of 1-7.5μg/mL (P<0.01). IC50 for SMMC-7721 cells (1.68 nmol/L) was lower than that for L02 cells (5.68 nmol/L), showing a preferential cytotoxicity of rHDL-ACM for SMMC-7721 cells. CONCLUSION: rHDL-ACM complex keeps the basic physical and biological binding properties of native HDL and shows a preferential cytotoxicity for SMMC-7721 hepatoma to normal L02 hepatocytes, HDL is a potential carrier for delivering lipophilic antitumoral drug to hepatoma cells.

Time-and pH-dependent colon-specific drug delivery for orally administered diclofenac sodium and 5-aminosalicylic acid

AIM: To investigate Time- and pH-dependent colon-specific drug delivery systems (CDDS) for orally administered diclofenac sodium (DS) and 5-aminosalicylic acid (5-ASA), respectively. METHODS: DS tablets and 5-ASA pellets were coated by ethylcellulose (EC) and methacrylic acid copolymers (Eudragit L100 and $100), respectively. The in vitro release behavior of the DS coated tablets and 5-ASA coated pellets were examined, and then in vivo absorption kinetics of DS coated tablets in dogs were further studied. RESULTS: Release profile of time-dependent DS coated tablets was not influenced by pH of the dissolution medium, but the lag time of DS release was primarily controlled by the thickness of the coating layer. The thicker the coating layer, the longer the lag time of DS release is. On the contrary, in view of the pH-dependent 5-ASA coated pellets, 5-ASA release was significantly governed by pH. Moreover, the 5-ASA release features from the coated pellets depended upon both the combination ratio of the Eudragit~ L100 and S100 pH-sensitive copolymers in the coating formulation and the thickness of the coating layer. The absorption kinetic studies of the DS coated tablets in dogs demonstrated that in vivo lag time of absorption was in a good agreement with in vitro lag time of release. CONCLUSION: Two types of CDDS, prepared herein by means of the regular coating technique, are able to achieve site-specific drug delivery targeting at colon following oral administration, and provide a promising strategy to control drug release targeting the desired lower gastrointestinal region.

Anti-hepatocarcinoma effects of 5-fluorouracil encapsulated by galactosylceramide liposomes in vivo and in vitro

AIM:To study the anti-hepatocarcinoma effects of 5-fluorouracil (5-Fu) encapsulated by galactosylceramide liposomes (5-Fu-GCL) in vivo and in vitro. METHODS: Tumor-bearing animal model and HepA cell line were respectively adopted to evaluate the anti-tumor effects of 5-Fu-GCL in vivo and in vitro. Tumor cell growth inhibition effects of 5-Fu-GCL in vitro were assessed by cell viability assay and MTT assay. In vivo experiment, the inhibitory effects on tumor growth were evaluated by tumor inhibition rate and animal survival days. High performance liquid chromatography was used to detect the concentration-time course of 5-Fu-GCL in intracellular fluid in vitro and the distribution of 5-Fu-GCL in liver tumor tissues in vivo. Apoptosis and cell cycle of tumor cells were demonstrated by flow cytometry. RESULTS: In vitro experiment, 5-Fu-GCL (6.25-100 μmol/L) and free 5-Fu significantly inhibited HepA cell growth. Furthermore, IC50 of 5-Fu-GCL (34.5 μmol/L) was lower than that of free 5-Fu (51.2 μmol/L). In vivo experiment, 5-Fu-GCL (20, 40, 80 mg/kg) significantly suppressed the tumor growth in HepA bearing mice model. Compared with free 5-Fu, the area under curve of 5-Fu-GCL in intracellular fluid increased 2.6 times. Similarly, the distribution of 5-Fu-GCL in liver tumor tissues was significantly higher than that of free 5-Fu. After being treated with 5-Fu-GCL, the apoptotic rate and the proportion of HepA cells in the S phase increased, while the proportion in the G0/G1 and G2/M phases decreased. CONCLUSION: 5-Fu-GCL appears to have anti-hepatocarcinoma effects and its drug action is better than free 5-Fu. Its mechanism is partly related to increased drug concentrations in intracellular fluid and liver tumor tissues, enhanced tumor cell apoptotic rate and arrest of cell cycle in S phase.

Gastroprotective effect and mechanism of amtolmetin guacyl in mice

AIM: To investigate the gastroprotective effect and mechanism of amtolmetin guacyl (AMG, MED15) in mice.METHODS: Male and female Kunming strain mice,weighing 18-22 g, were utilized in the experiment. Normal or ethanol-induced gastric mucosal damage models in mice were successfully established to investigate the gastroprotective effect and mechanism of AMG. In the experiment of gastric mucosal damage after repeated treatment with AMG, the mice were randomly divided into 5 groups: normal group, 3 AMG groups receiving (75, 150 and 300 mg/kg), and tolmetin group receiving 90 mg/kg.The mice were randomly divided into 6 groups as follows:normal group, model group, AMG groups with doses of 75, 150 and 300 mg/kg, respectively, and tolmetin group with a dose of 90 mg/kg in ethanol-induced gastric mucosal damage experiment. The severity of gastric mucosal lesions was scored from 0 to 5. Gastric tissue sections were stained with hematoxylin and eosin (HE) and examined under light microscopy. Also gastric tissue sections were stained with uranyl acetate and lead citrate, and examined under electron microscopy. In addition, nitric oxide (NO) and malondialdehyde (MDA) contents, and nitric oxide synthase (NOS) and superoxide dismutase (SOD) activities in the stomach tissue homogenates were measured by biochemical methods.RESULTS: Repeated treatment with AMG (75, 150 and3 00 mg/kg) for 7 d did not induce any appreciable mucosal damage, and the average score was not significantly different from that of normal mice. In contrast, tolmetin (90 mg/kg) produced significant gastric mucosal lesions compared with the normal group (P<0.01). AMG (75, 150 and 300 mg/kg) significantly reduced the severity of gastric lesions induced by ethanol in a dose-dependent manner as compared with the model group (P<0.05, AMG 75 and 150 mg/kg vs model; P<0.01, AMG 300 mg/kg vs model).Light and electron microscopy revealed that AMG (150 and 300 mg/kg) induced minimal changes in the surface epithelium layer, without vascular congestion or leucocyte adherence. AMG (75,150 and 300 mg/kg) demonstrated dose-dependent gastroprotective effects on mice in ourstudy. AMG (75, 150 and 300 mg/kg) could significantly increase NO content and NOS level in the stomach homogenates of mice compared with the model group (P<0.05, AMG 75 mg/kg and 150 mg/kg groups vs model group; P<0.01, AMG 300 mg/kg vs model group) respectively. Moreover, AMG (150 and 300 mg/kg) not only significantly increased SOD activities but also obviously decreased the MDA content in the stomach homogenates of mice.CONCLUSION: AMG exerts significant gastroprotective actions on mice and the involved mechanisms may be its antioxidative effect and induction of NO production.

A New Cyclic Bisdesmoside from Tubers of Bolbostemma paniculatum

The structure of tubeimoside V (1), a new cyclic bisdesmoside, isolated from tubers of Bolbostemma paniculatum (Tu Bei Mu), was established by means of 2D NMR spectral and chemical methods. Compound 1 has inter-saccharide chain bridging by a dicrotalic acid to form a unique macrocyclic structure.

New Metabolites of Aconitine in Rabbit Urine

A sensitive analytical method to identify and determine aconitine and its metabolites in rabbit urine was developed by liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MSn).In this method,aconitine and its four metabolites in rabbit urine were isolated and deduced As 16-O-demethylaconine(M1), benzoylaconine(M2), 16-O-demethylbenzoylaconine (M3)and aconine(M4).M1 and M3 are new metabolites of aconitine and M2 and M4 are first identified in rabbit urine.

Inhibitory effect of heparin-derived oligosaccharides on secretion of interleukin-4 and interleukin-5 from human peripheral blood T lymphocytes

AIM: To investigate the inhibitory effect of heparin-derived oligosaccharides (Oligs) on secretion of interleukin-4 (IL-4) and interleukin-5 (IL-5) from human peripheral blood T lymphocytes (PBTLs).METHODS: Oligs were prepared by three different heparin depolymerization methods and separated by gel filtration chromatography. PBTLs from ten adult patients with allergic eosinophilic gastroenteritis were treated with phytahematoagglutinin (PHA) and Oligs. The supernatants from the cell culture of PBTLs were harvested and subjected to the determination of IL-4 and IL-5 contents by ELISA method.RESULTS: At the concentration of 5μg/mL, Oligs with different Mr had different effects on the secretion of IL-4 and IL-5. The tetrasaccharide with Mr of 1 142, produced by depolymerizing heparin with hydrogen peroxide, had the strongest inhibitory effect on the secretion of IL-4. It decreased the IL-4 content from 375.6±39.2 ng/L (PHA group) to 12.5±5.7 ng/L (P<0.01). The hexasaccharide with Mr of I 806, produced by depolymerizing heparin with βelimination method, had the strongest inhibitory effect on the secretion of IL-5. It decreased the IL-5 content from 289.2±33.4 ng/L (PHA group) to 22.0±5.2 ng/L (P<0.01).CONCLUSION: The inhibitory activity of Oligs on the secretion of IL-4 and IL-5 from human PBTLs closely depends on their molecular structure, and there may be an essential structure to act as an inhibitor. The most effective inhibitors of IL-4 and IL-5 secretion are tetrasaccharides and hexasaccharides, respectively.

A New Asterosaponin from the Starfish Culcita novaeguineae

A new asterosaponin named novaeguinoside A, along with a known saponin, asteronyl pentaglycoside sulfate, was isolated from the starfish Culcita novaeguineae. The new compound was identified to be sodium 6α-O-{β-D-fucopyranosyl-(1→2)-β-D-fucopyranosyl-(1→4)-[β-D- quinovopyranosyl-(1→2)]-β-D-xylopyranosyl-(1→3)-β-D-quinovopyranosyl}-5α-pregn-9(11)-en- 20-one-3β-yl-sulfate by extensive spectral analysis and chemical evidence.

Desmosdumotin A, a New Compound from Desmos dumosus

A new compound, desmosdumotin A (1), was isolated from the roots of Desmos dumosus (Roxb.) Saff. Its chemical structure was established as 5-hydroxy-7-methoxy-8-formyl-3-benzoyl-2,6-dimethyl-2R,3S-dihy-drochromone on the basis of spectral methods, including ^1H-^1H COSY, ^1H-^13C COSY, and HMBC as well as single-crystal X-ray analysis.

Determination of Five Organic Acids in Radix Isatidis by Column Partition Chromatography and Capillary Zone Electrophoresis柱分配色谱-毛细管区带电泳联用分析板蓝根中五种微量有机酸(英文)

Aim To determine five organic acids in Radix Isatidis . Method The extraction method and the column partition chromatographic conditions were studied. Then a capillary zone electrophoretic method was set up for the determination. Results The linear ranges of quinazolinone acid, n anthranilic acid, benzoic acid, salicylic acid, and syringic acid were 5 52-92 0 μg·mL -1 , 5 12-102 μg·mL -1 , 2 28-84 4 μg·mL-1 , 4 78-159 μg·mL -1 , and 1 74-87 0 μg·mL -1 respectively. Conclusion The established method is accurate and simple.

High-Performance Liquid Chromatographic Method for Determination of Germacrone in Rat Plasma

Aim A reliable high-performance liquid chromatographic (HPLC) method wasdeveloped for determination of germacrone in rat plasma. Methods The plasma samples were treatedwith acetonitrile and analyzed by HPLC with UV detection at 244 nm. Results The limit of detectionwas 100 ng·mL^(-1) for germacrone in plasma and the linear range was 0.1004-15.06 μg·mL^(-1) inplasma. The RSD of intra-day and inter-day assay was 1.87% - 4.29% and 1.29% -5.15%, respectively.The recoveries of germacrone were over 95%. The endogenous substances in plasma did not show anyinterference in the analysis. Conclusion The method is accurate and convenient, and suitable forpharmacokinetic studies of germacrone in rats.

Improving Bioavailability of Naftopidil in Dogs by Using Bioadhesion

To improve the bioavailability of naftopidil, bioadhesive sustained-release capsules and nonbioadhesive capsules were prepared. Bioadhesive polymers such as hydroxypropyl methylcellulose (HPMC) and Carbopol 934 (CP 934) were used in the bioadhesive capsules formulations. Naftopidil capsule and two formulations of bioadhesive sustained-release capsules (I and II) were respectively given to five healthy male dogs in an open randomized cross-over test. The naftopidil concentrations in plasma were determined by a newly developed HPLC method. The pharmacokinetic parameters and the relative bioavailability were measured. The AUC0→24, Cmax and Tmax,of non-bioadhesive naftopidil capsules were 3494.7±466.47 h.ng.mL^-1, 697.48±94.22 ng-mL^-1 and 1.15±40.49 h. These pharmacokinetic parameters of bioadhesive sustained-release capsules I and II were 4618.46±316.68 h-ng-mL^-1 and 4746.44±317.22 h.ng.mL^-1, 468.59±61.25 ng-mL^-1 and 512.00±72.29 ng.mL^-1, both 4.0±0.71 h respectively. Results from statistical analysis showed that there were significant differences between the two bioadhesive formulations and the non-bioadhesive one in AUC0→24, Cmax and Tmax The relative bioavailability of the two bioadhesive sustainedrelease capsules were respectively 133.40±12.72% and 137.53±17.49% when compared with non-bioadhesive capsules. The bioavailability of naftopidil in dogs was improved hy using bioadhesion.

A New Nitro Alkaloid from Corydalis saxicola Bunting

A new nitro tetrahydronprotoberberins alkaloid, 1-nitro-apocavidine was isolated from Corydalis saxicola Bunting. The structure was established by spectroscopic methods.

Arterial baroreflex function determines the survival time in lipopolysaccharide-induced shock in rats

Lipopolysaccharide(LPS) mimics many of the effects of septic shock. LPS-induced death has been attributed to systemic hypotension, hyporeactiveness to vasoconstrictors, metabolic acidosis.and organ damage. However, there is no research directed to the involvement of the baroreflex sensitivity (BRS) in LPS-induced death. The purpose of this study was to evaluate the effect of BRS on the survival time after lethal LPS challenge.

A New Iridoid Glucoside from Lagotis yunnanensis

Phytochemical investigation of Lagotis yunnanensis led to the isolation and identification of a new iridoid glucoside 1, named as 10-O-(3,4-dimethoxy-(E)-cinnamoyl)aucubin. Its structure was elucidated by spectroscopie methods.

A New Bisxanthone from Hypericum japonicum Thunb.ex Murray

A new bisxanthone, named bijaponicaxanthone C, was isolated from the whole plant of Hypericum japonicum. The structure was elucidated as 6-[1’’,5’’,6’’-trihydroxy-2’’’-(β-hydroxy-β- methylethyl)-2’’’,3’’’-dihydrofuran(5’’’,4’’’,3’’,4’’)xanthone-3’’’-oxyl]-1,3,5-trihydroxy-4-isoprenylxant- hone (1) on the basis of the spectral and chemical evidences.

Enantiomeric Separation of Meptazinol Hydrochloride by Capillary Electrophoresis

Aim To establish a capillary electrophoresis method for enantiomericseparation of meptazinol hydrochlo-ride. Methods The separation conditions such ascyclodextrin(CD)type, buffer pH, concentration of 2,3,6-O-trimethyl-β-cyclodextrin and organicadditives were optimized. An optimum concentration was 30 mmol· L^(-1) phosphate (pH 7.02) with 10%(W/V) TM-β-CD and 2% acetonitrile. Results Baseline resolution of the enantiomer was readilyachieved using 2, 3, 6-O-trimethyl-β-cyclodextrin. Conclusion This is a convenient method for fastenantiomeric resolution of meptazinol hydrochloride.