12-O-acetylphorbol-13-decanoate potently inhibits cytopathic effects of human immunodeficiency virus type 1 (HIV-1), without activation of protein kinase C.

Through bioactivity-guided fractionation, eight phorbol diesters, including five new ones (1-5), were isolated from the seeds of Croton tiglium collected in Egypt. 12-O-Acetylphorbol-13-decanoate (6) and 12-O-decanoylphorbol-13-(2-methylbutyrate) (4) potently inhibited the HIV-1-induced cytopathic effect on MT-4 cells (IC100 values of 7.6 ng/ml and 7.81 micrograms/ml, and CC0 values of 62.5 micrograms/ml and 31.3 micrograms/ml, respectively) without activating protein kinase C.     

Effects of isoflavone compounds on the activation of phospholipase C.

The effect of isoflavone compounds, genistein and daidzein, on the breakdown of inositol phospholipids in 3T3 cells was studied. Genistein (100 micrograms/ml) inhibited the stimulation of the production of inositol phosphates by bombesin. The stimulated production of inositol phosphates by AlF-4 was also inhibited by genistein (IC50 = 0.6 micrograms/ml) and daidzein (IC50 = 2 micrograms/ml). However, the catalytic activity of phospholipase-C (PLC) in 3T3 cell extracts was not inhibited by these isoflavones. These results suggest that the isoflavones inhibited the activation of PLC at the G-protein or downstream of the sequences in signal transduction. In permeabilized 3T3 cells, the inhibition of AlF-4 plus adenosine triphosphate (ATP)-dependent PLC was recovered by increasing ATP but not AlF-4. Genistein also inhibited the activity of adenosine 5'-[3-O-thiotriphosphate] (ATP[S])-dependent PLC. The effect of genistein and other inhibitors of protein tyrosine kinases and phosphatases suggests that protein tyrosine phosphorylation is not involved in the activation of PLC in 3T3 cells and that AlF-4- and ATP[S]-mediated activation of PLC involves a different mechanism from the tyrosine kinase-mediated activation of PLC. Daidzein and genistein seem to interrupt the ATP-dependent step of PLC activation by a putative G-protein.     

A stimulatory effect of Artemisia leaf extract on the proliferation of cultured endothelial cells

To investigate the effect of the hot water extract from Artemisia leaf (Artemisia princeps Panpanini) (AFE) on the proliferation of endothelial cells, the cells from bovine aorta were cultured for up to 96 h in the presence of 1, 5, 10 or 50 micrograms/ml AFE in RPMI1640 medium supplemented with 10% fetal bovine serum. After a 72 h culture, the cell number was significantly increased by AFE at 1, 5 and 10 micrograms/ml. An increase in the cell number by 5 micrograms/ml AFE observed after a 72 or 96 h treatment. The incorporations of both [3H]thymidine and [14C]leucine by the growing cells were significantly increased by 5 micrograms/ml AFE after a 72 h treatment. In addition, the incorporation of [3H]thymidine by either growing or confluent cells was significantly increased by 50 micrograms/ml AFE after a 72 h treatment. The stimulatory activity of AFE was recognized in the low-molecular-weight fraction (molecular weight less than or equal to 10000 dalton). These results clearly indicated that AFE contained some low-molecular-weight component(s) which stimulates the proliferation of vascular endothelial cells in vitro.     

Enteric solid dispersion of ciclosporin A (CiA) having potential to deliver CiA into lymphatics

Solid dispersions composed of three components, ciclosporin A (CiA), surfactant (HCO-60) and a pharmaceutical additive, were prepared. As an additive, cellulose acetate phthalate (CAP), methacrylic acid and methacrylic acid methylester copolymer (Eudragit L-100) and hydroxypropylmethylcellulose phthalate (HP-55), which are generally used as enteric coating materials, were employed. The dissolution behavior of CiA from these enteric solid-dispersion system was studied according to the paddle method of JP XI in comparison with that of Sandimmun, an olive oily CiA solution as a reference. Solid dispersion of CiA preparation did not dissolve in the 1st test fluid (pH 1.2) in 2 h. In the 2nd fluid (pH 6.8), about 80% of CiA was dissolved within 12 min, though the dissolution rate was dependent on both the quality and quantity of the additives. An in vivo systemic and lymphatic availability study was performed with rats whose carotid artery and thoracic lymph duct were cannulated. After intrastomach administration of each CiA preparation to rats at a dose of 7 mg/kg, blood and lymph samples were collected for 6 h. One of the HP-55 preparations gave the highest plasma CiA level, Cmax = 0.99 +/- 0.20 (S.E., n = 4) micrograms/ml, and also showed the highest lymphatic availability, the percentage of dose delivered to the lymphatics in 6 h was 1.98 +/- 0.10% and the maximum lymph CiA level was 76.8 +/- 12.86 micrograms/ml. Lymphatic availability of CiA from Sandimmun was 0.78 +/- 0.11% and the peak plasma CiA level was 0.46 +/- 0.10 microgram/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

The biological function of rabbit blastocyst peptides (RBPs)

The inhibitory effect of rabbit blastocystic peptides (RBPs) on lymphocyte transformation was studied by the method of measuring the incorporation of 3H-thymidine into DNA. The results indicated that RBPs inhibited PHA-stimulated rat and human lymphocyte transformation in vitro. In the concentration ranging from 40 to 200 micrograms/2.5 ml, the inhibition was dose-dependent. No obvious inhibitory action was found with hCG (16-128) IU/2.5 ml), progesterone (250 ng-1 microgram/2.5 ml) and pregnant rabbit serum. It was further demonstrated that RBPs at a higher dosage (200 micrograms/ml) was inhibitory to PGF2 alpha secretion. On the other hand, the 3H-leucine incorporation of rabbit endometrium was enhanced by these peptides, and this action could be blocked by the addition of actidine.     

Anti-plasmin inhibitor. VI. Structure of phlorofucofuroeckol A, a novel phlorotannin with both dibenzo-1,4-dioxin and dibenzofuran elements, from Ecklonia kurome Okamura

Phlorofucofuroeckol A (1), a novel phlorotannin with both dibenzo-1,4-dioxin and dibenzofuran elements, has been isolated from the brown alga Ecklonia kurome OKAMURA as a potent anti-plasmin inhibitor. Its structure has been elucidated to be 1,11-di(3,5-dihydroxyphenoxy)- benzofuro[3,2-a]dibenzo[b,e][1,4]dioxin-2,4,8,10,14-pentaol on the basis of the spectral data, in particular, by means of negative nuclear Overhauser effect (NOE) and long-range carbon-proton couplings (2JCH and 3JCH). Phlorofucofuroeckol A inhibited the action of alpha 2-macroglobulin (IC50 = 1.0 micrograms/ml) and alpha 2-plasmin inhibitor (IC50 = 0.3 micrograms/ml), the main plasmin inhibitors in plasma.     

Determination of cholesterol and cortisone absorption in polyurethane. I. Methodology using size-exclusion chromatography and dual detection.

A size-exclusion chromatographic method is described for measuring the absorption of the steroid-based lipids cholesterol and cortisone into Pellethane 2363, a polyurethane used in biomedical implants. The method uses refractometry and ultraviolet diode-array detection, with tetrahydrofuran as the mobile phase. Using an injection volume of 150 microliters, the lower limit of accurate measurement for cholesterol (refractive index detection) was 6 micrograms/ml with a lower limit of detection, based on a 2:1 signal-to-noise ratio, of 0.15 micrograms (1 microgram/ml). For cortisone (ultraviolet detection), the lower accurate limit was 0.6 micrograms/ml with a lower limit of 0.015 micrograms (0.1 micrograms/ml). The results show that after 44 h, 2037 micrograms/g cholesterol and 3131 micrograms/g cortisone were absorbed by the polyurethane. The method eliminates extensive sample manipulation and is sensitive to low levels of lipid in the presence of a high-molecular-mass synthetic polymer.     

Gas chromatographic assay for the new antitumor agent sulfamic acid diester (NSC 329680) and its stability in buffer, blood and plasma

A sensitive gas chromatographic assay with electron-capture detection has been developed for sulfamic acid diester (sulfamic acid 1,7-heptanediyl ester, NSC 329680) based on its conversion to 1,7-diiodoheptane in the presence of excess sodium iodide. The assay is linear up to 1 microgram/ml sulfamic acid diester and has a lower limit of detection of 25 ng/ml from 0.5 ml plasma. The coefficient of variation of the assay is 6.4% at 1 microgram/ml and 8.0% at 100 ng/ml. Sulfamic acid diester is relatively stable in 0.9% sodium chloride and 0.1 M sodium phosphate buffers, pH 7.0-9.0, with half-lives greater than 38 h. The major breakdown product of sulfamic acid diester is sulfamic acid 1,7-heptane-monoyl ester. When added to whole blood sulfamic acid diester shows concentration-dependent breakdown. At 50 and 100 micrograms/ml sulfamic acid diester, the half-time in whole blood is 6.9 h and 65% of the drug is sequestered by the blood cells. At 10 micrograms/ml sulfamic acid diester in blood, there is no detectable breakdown of the drug over 24 h and all of the drug is sequestered by the blood cells. Protein binding of sulfamic acid diester in human plasma is 82% at 10 micrograms/ml and 68% at 100 micrograms/ml.     

Diverted total synthesis of melodorinol analogues and evaluation of their cytotoxicity

A series of melodorinol analogues were synthesized via a diverted total synthesis approach, leading to structural modifications on several regions of the molecule. Their cytotoxicity was evaluated against five human cancer cell lines (KB, HeLa-S3, MCF-7, HT-29 and A549). Structure-activity relationship studies revealed key parameters that affect the cytotoxicity. In particular, the novel 4-bromo-furanone analogues exhibited greater cytotoxicity compared to the corresponding non-brominated analogues. The stereochemistry at C-6 and the nature of acyl substituents on the C-6 and C-7 hydroxyl groups also play an important role. The most potent analogues exhibit approximately 15-fold higher cytotoxicity towards KB and HeLa-S3 than melodorinol and also show exceptionally high potency against MCF-7, HT-29 and A549 cell lines.     

Synthesis of (±)-cyclic dehypoxanthine futalosine, the biosynthetic intermediate in an alternative biosynthetic pathway for menaquinones

The first synthesis of (±)-cyclic dehypoxanthine futalosine (cyclic DHFL), a biosynthetic intermediate in the futalosine pathway for menaquinones operating in microorganisms, has been achieved. Efficient growth of the Streptomyces coelicolor mutant, which lacks the cyclic DHFL synthetase gene (mqnC gene) was observed in the presence of synthetic (±)-cyclic DHFL.     

Simultaneous determination of tranilast and metabolites in plasma and urine using high-performance liquid chromatography

A method has been developed for the simultaneous determination of Tranilast, N-(3',4'-dimethoxycinnamoyl)anthranilic acid (N-5'), and metabolites in plasma and urine from humans, dogs and rodents administered N-5'. Total N-5' and metabolite N-3 conjugates were determined in human urine. Detection limits in plasma were 0.2 micrograms/ml for metabolite N-3-S and N-5' and 0.1 micrograms/ml for metabolites N-3 and N-4. In urine, detection limits were 2 micrograms/ml for metabolite N-3-S and N-5' and 1 micrograms/ml for metabolites N-3 and N-4. Metabolite N-4 was not identified in any sample assayed.     

Flow-injection chemiluminometric determination of some thioxanthene derivatives in pharmaceutical formulations and biological fluids using the [Ru(dipy)3(2+)]-Ce(IV) system.

A flow-injection (FI) methodology using tris(2,2'-dipyridyl)ruthenium(II), [Ru(dipy)3(2+)], chemiluminescence (CL) was developed for the rapid and sensitive determination of three thioxanthene derivatives, namely zuclopenthixol hydrochloride, flupentixol hydrochloride and thiothixene. The method is based on the CL reaction of the studied thioxanthenes with [Ru(dipy)3(2+)] and Ce(IV) in a sulfuric acid medium. Under the optimum conditions, calibration graphs were obtained over the concentration ranges 0.002-6 migrograms/ml for zuclopenthixol hydrochloride, 0.5-15 micrograms/ml for flupentixol hydrochloride and 0.05-7.5 micrograms/ml for thiothixene. The limits of detection (s/n = 3) were 4.2 x 10(-9) mol/l zuclopenthixol hydrochloride, 2 x 10(-8) mol/l flupentixol hydrochloride and 4.5 x 10(-8) mol/l thiothixene. The method was successfully applied to the determination of these compounds in dosage forms and biological fluids.     

Incarnal. A new antibacterial sesquiterpene from Basidiomycetes

A new sesquiterpene, incarnal, was isolated from culture fluid of Gloeostereum incarnatum (Japanese name: Nikawaurokotake). Incarnal inhibited the growth of gram-positive bacteria at 6.25-12.5 micrograms/ml. The structure of incarnal has been determined to be (1-hydroxy-2,10,10-trimethyl)-3-methylene- tricyclo[6.3.0.0(2.6)]undec-5,7-diene-4,9-dione by X-ray diffraction and spectroscopic methods.     

Effects of saikosaponin metabolites on the hemolysis of red blood cells and their adsorbability on the cell membrane

The hemolytic properties and the adsorbability on red blood cells of saikosaponin a, saikosaponin d and 13 metabolites formed in the alimentary tract were investigated. Among these compounds, saikosaponin d and its intestinal product, prosaikogenin G, which possess an alpha-hydroxyl function at C16, showed the strongest hemolytic activity at the dose range of 1.0 to 5.0 micrograms/ml. Saikosaponin a and its intestinal product, prosaikogenin F, which possess a beta-hydroxyl function at C16, showed activity above 10 micrograms/ml. In this case, the monoglycoside, prosaikogenin F, showed the stronger activity than the diglycoside, saikosaponin a. Among the gastric products whose ether ring was cleaved to produce a carbinol, the monoglycosides, prosaikogenin A and prosaikogenin H, showed a slight activity above 25 micrograms/ml, and the saikogenins except saikogenin A were inactive. Saikogenin A, however, had hemolytic activity at a dose of 15 micrograms/ml. The adsorbabilities of these compounds on red blood cell membranes closely paralleled their degrees of hemolytic activity.     

Determination of inorganic anions by ion chromatography using a graphitized carbon column dynamically coated with cetyltrimethylammonium ions.

The simultaneous determination of inorganic anions by ion chromatography using a dynamically coated graphitized carbon column with cetyltrimethylammonium (CTA) ions was investigated with suppressed conductivity detection. Column preparations with CTA and sodium carbonate-sodium hydrogencarbonate concentration in the eluent were examined to optimize the separation of seven common anions (F-, Cl-, NO2-, Br-, NO3-, HPO(4)2- and SO(4)2-). Calibration curves were linear from 0.5 to 5 micrograms/ml for F-, from 1.0 to 10 micrograms/ml for Cl-, from 1.5 to 15 micrograms/ml for NO2-, from 2.0 to 20 micrograms/ml for Br- and NO3-, from 5.0 to 50 micrograms/ml for HPO(4)2- and from 3.0 to 30 micrograms/ml for SO(4)2- with correlation coefficients (r) of 0.999 or better. The relative standard deviations of peak areas were between 0.3 and 0.9% for 10 repeated measurements. The application of this newly developed method was demonstrated by the determination of inorganic anions in the water for pharmaceutical purposes. Using CTA-Br as the coating agent, a permanently coated ion-exchange column was obtained, which allowed efficient separations of seven anions without adding any coating agent to the eluent.     

Pharmacokinetics of [6]-gingerol after intravenous administration in rats

A high-performance liquid chromatographic method to determine [6]-gingerol, a pungent constituent of ginger, in rat plasma was developed and a pharmacokinetic study was performed in rats. Quantitative analysis with high reproducibility was achieved for [6]-gingerol over the concentration range of 0.2-40 micrograms/ml. After bolus intravenous administration at a dose of 3 mg/kg, the plasma concentration-time curve was described by a two-compartment open model. [6]-Gingerol was rapidly cleared from plasma with a terminal half-life of 7.23 min and a total body clearance of 16.8 ml/min/kg. Serum protein binding of [6]-gingerol was 92.4%.     

Biopolymers from marine invertebrates. XII. A novel cytolytic factor from a hermit crab, Clibanarius longitarsus.

Bioactive polymers were sought in marine arthropoda and a novel cytolytic factor was found in a hermit crab, Clibanarius longitarsus. The partially purified factor showed activity in fractions corresponding to a molecular weight of about 10 kilodaltons on a Sephadex G-75 column. This cytolytic factor was halfmaximally active for tumor cells at 0.13-0.66 micrograms/ml and for normal cells at 1.9-82 micrograms/ml. Tumor lysis by the factor was time dependent and was complete within 12 h. This bioactive polymer was labile on heating, at low and high pH.     

Determination of ceftriaxone, a novel cephalosporin, in plasma, urine and saliva by high-performance liquid chromatography on an NH2 bonded-phase column

A high-performance liquid chromatographic method has been developed for the determination of a new cephalosporin antibiotic in plasma, urine and saliva (mixed saliva) using normal-phase technique and an NH2 bonded-phase column. The eluent mixture was a combination of acetonitrile and an aqueous solution of ammonium carbonate. The rapid method involved precipitation of protein from fluids by means of acetonitrile followed by automatic injection of the supernatant. The detection limit was 0.4 micrograms/ml for plasma, 3 micrograms/ml for urine and 0.03 micrograms/ml for saliva using UV detection.