Detection of the phosphorothioate oligonucleotide fomivirsen using a ligase detection reaction with polymerase chain reaction.

This study aimed to develop a simple and sensitive detection method for fomivirsen, a 21-nucleotide phosphorothioate oligonucleotide used as a nucleic acid medicine, using a ligase detection reaction. A ligation probe was designed to hybridize with fomivirsen and polymerase chain reaction (PCR) primers, with a deoxyuridine part between the primer binding sites. The probe was ligated to a circular product by Taq DNA ligase, and the resulting product was converted to a linear form through the removal of the uracil base using uracil DNA glycosylase. The linear product was then quantified using real-time PCR. The developed method could detect 0.025-6.4 nM of fomivirsen in water and HeLa genomic DNA solutions and 0.6-160 nM of fomivirsen in mouse serum in combination with an extraction method based on alkalinization and neutralization. This method could be useful for not only detecting fomivirsen but also other functional oligonucleotides composed of phosphorothioate oligonucleotides. In summary, this study presents a practical and effective approach to the detection of the nucleic acid medicine fomivirsen.

Relationship Between Amplicon Size and Heat Conditions in Polymerase Chain Reaction Detection of DNA Degraded by Autoclaving.

This study examined the influence of heat exposure on DNA samples during polymerase chain reaction (PCR) detection. In this study, λDNA samples, as model DNA, were exposed to 105°C for 3-90 minutes or to 105°C-115°C for 15 minutes by autoclaving. The exposed samples were subjected to real-time PCR using nine primer sets with amplicon sizes of 45-504 bp. Regarding DNA samples exposed to 105°C by autoclaving, the data showed negative correlations between the logarithm of λDNA concentration (log λDNA) calculated using real-time PCR and exposure duration and a good relationship between the slope of the regression line and amplicon size. Regarding λDNA samples exposed to heat for 15 minutes, the data showed negative correlations between the log λDNA and exposure temperature and a good relationship between the slope of the regression line and amplicon size. These results showed that the equations used in this study could predict the degree of degradation in λDNA samples by autoclaving, and the PCR detection levels of the DNA at each amplicon size.

Real-time PCR method for detection of short DNA using a deoxyuridine probe and application for detection of fomivirsen.

This study sought to develop a short DNA detection method using a deoxyuridine probe and polymerase chain reaction. The probe was hybridized to the target short DNA, which was then extended by DNA polymerase. The extended DNA was used for real-time PCR after the probe was removed by uracil DNA glycosylase. This method measured from 0.01 to 10 nM of a model short DNA sequence of 17 nucleotides. The method was then used to detect the nucleic acid medicine fomivirsen, as well as 21 phosphorothioate nucleotides, and to quantify 0.1-100 nM of fomivirsen. This method may be useful for detecting short DNA fragments, such as functional nucleotides.

Complanadine F, a novel dimeric alkaloid from Lycopodium complanatum.

A novel Lycopodium alkaloid, complanadine F (1), seco-complanadine A type was isolated from the club moss Lycopodium complanatum. The planar structure and relative configuration were elucidated based on spectroscopic data.

Partition Efficiency of Eccentric Coil for Countercurrent Chromatographic Separation of Proteins Using Small-scale Cross-axis Coil Planet Centrifuge with Circular and Elliptic Cylindrical Columns.

The partition efficiency of the double-spaced coil for eccentric and toroidal coils on countercurrent chromatographic separation of proteins was evaluated using the small-scale cross-axis coil planet centrifuge (CPC) equipped with circular and elliptic cylindrical columns. Standard cytochrome c, myoglobin and lysozyme samples were used for separation with the 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate system. In the circular column, the double-spaced eccentric coil yielded better peak resolution than the double-spaced toroidal coil, and the double-spaced eccentric coil yielded better peak resolution than the single-spaced eccentric coil. In the elliptic column, the double-spaced eccentric coil also produced better peak resolution than the double-spaced toroidal coil, but the single-spaced eccentric coil yielded better peak resolution than the double-spaced eccentric coil. The overall results indicated that the double-spaced eccentric coil for the circular column and the single-spaced eccentric coil for the elliptic column yielded better protein separation using the small-scale cross-axis CPC with aqueous two-phase solvent systems.

Partition efficiencies of three different coiled columns for the coil satellite centrifuge at higher rotation speed combinations.

The partition efficiencies of three different coiled columns, conventional multilayer coiled column, eccentric coiled column and toroidal coiled column, were evaluated by the separation of 4-methylumbelliferyl sugar derivatives using the coil satellite centrifuge (CSC) with an organic-aqueous two-phase solvent system composed of ethyl acetate/1-butanol/water. The CSC apparatus was reinforced the planet axis to maintain the stable satellite motion, which was completed by combining the rotation of three axes including the sun axis (the angular velocity, ω1), the planet axis (ω2) and the satellite axis (ω3) under the relation at ω1 = ω2 + ω3. In the present study, four different rotation speed combination types were used for the separation at the ratio (ω1, ω2, ω3) = I. (300, 150, 150), II. (300, 100, 200), III. (300, 147, 153) and IV. (300, 200, 100 rpm) under different revolution speeds of ω1 = 300, 400 and 500 rpm. In the conventional multilayer coiled column with the upper mobile phase, the rotation speed combination type II yielded the best peak resolution while the rotation speed combination types III and IV had extremely low stationary phase retention even at higher revolution speeds. This inconvenience was eliminated by using the eccentric and the toroidal coiled column. The rotation speed combination type II for the eccentric coiled column and the type IV for the toroidal coiled column produced the best separation in both the upper and the lower mobile phases among four different rotation speed combination types. The overall results indicated that better peak resolution was obtained by the eccentric coiled column than by the toroidal coiled column except for the separation with the upper mobile phase at the low revolution speed.

Kinetic Study of the Equilibration between Carminic Acid and Its Two Isomers Isolated from Cochineal Dye.

Carminic acid (CA) is a major component of cochineal dye used in food additives, cosmetics, and pharmaceuticals. CA and its isomers, 2-C-α-glucofuranoside and 2-C-ß-glucofuranoside of kermesic acid (DCIV and DCVII, respectively), were isolated from cochineal dye and the equilibrium constants (K) between CA, DCIV and DCVII were investigated. DCIV was partially converted to CA and DCVII, and DCVII was converted to CA and DCIV, whereas CA was very stable and only very slightly converted to DCIV and DCVII. Most of the DCIV and DCVII was converted to CA under aqueous conditions. The kinetic rate constants (k) for the degradation of DCIV within the first day of incubation at 24°C was determined to be 0.901 d-1 and for the degradation of DCVII it was determined to be 1.102 d-1. The k value for the formation of CA from the remaining DCIV was calculated to be 0.146 d-1 and for the formation of CA from the produced DCVII it was found to be 0.148 d-1. The K values were calculated as 1.22×10-7, 2.61×10-3 and 2.36×10-3 mol/L for CA, DCIV and DCVII, respectively. These findings will be helpful for ensuring the safety and for aiding the quality assurance of cochineal dye products.

Comparison of the peak resolution and the stationary phase retention between the satellite and the planetary motions using the coil satellite centrifuge with counter-current chromatographic separation of 4-methylumbelliferyl sugar derivatives.

Coil satellite centrifuge (CSC) produces the complex satellite motion consisting of the triplicate rotation of the coiled column around three axes including the sun axis (the angular velocity, ω1), the planet axis (ω2) and the satellite axis (the central axis of the column) (ω3) according to the following formula: ω1=ω2+ω3. Improved peak resolution in the separation of 4-methylumbelliferyl sugar derivatives was achieved using the conventional multilayer coiled columns with ethyl acetate/1-butanol/water (3: 2: 5, v/v) for the lower mobile phase at the combination of the rotation speeds (ω1, ω2, ω3)=(300, 150, 150rpm), and (1:4:5, v/v) for the upper mobile phase at (300:100:200rpm). The effect of the satellite motion on the peak resolution and the stationary phase retention was evaluated by each CSC separation with the different rotation speeds of ω2 and ω3 under the constant revolution speed at ω1=300rpm. With the lower mobile phase, almost constant peak resolution and stationary phase retention were yielded regardless of the change of ω2 and ω3, while with the upper mobile phase these two values were sensitively varied according to the different combination of ω2 and ω3. For example, when ω2=147 or 200rpm is used, no stationary phase was retained in the coiled column while ω2=150rpm could retain enough volume of stationary phase for separation. On the other hand, the combined rotation speeds at (ω1, ω2, ω3)=(300, 300, 0rpm) or (300, 0, 300rpm) produced insufficient peak resolution regardless of the choice of the mobile phase apparently due to the lack of rotation speed except at (300, 0, 300rpm) with the upper mobile phase. At lower rotation speed of ω1=300rpm, better peak resolution and stationary phase retention were obtained by the satellite motion (ω3) than by the planetary motion (ω2), or ω3>ω2. The effect of the hydrophobicity of the two-phase solvent systems on the stationary phase retention was further examined using the n-hexane/ethyl acetate/1-butanol/methanol/water system at different volume ratios. In the satellite motion at (ω1, ω2, ω3)=(300, 150, 150rpm), almost constant stationary phase retention was obtained with the lower mobile phase regardless of the hydrophobicity of the solvent system whereas the stationary phase retention varied according to the volume ratio of the two-phase solvent system for the upper mobile phase. However, stable stationary phase retention was observed with either phase used as the mobile phase. In order to analyze the acceleration acting on the coiled column, an acceleration sensor was set on the column holder by displacing the multilayer column. The combination of the rotation speeds at (300, 100, 200rpm) showed double loops in the acceleration track, whereas (300, 150, 150rpm) showed a single loop, and all other combinations showed, complex tracks. The overall results indicate that the satellite motion is seriously affected by the combination of rotation speeds and the hydrophobicity of the two-phase solvent system when the upper phase was used as the mobile phase for separation.

Improved Separations of Proteins and Sugar Derivatives Using the Small-Scale Cross-Axis Coil Planet Centrifuge with Locular Multilayer Coiled Columns.

1 BACKGROUND: Countercurrent chromatography (CCC) is liquid-liquid partition chromatography without using a solid support matrix. This technique requires further improvement of partition efficiency and shortening theseparation time. 2 METHODS: The locular multilayer coils modified with and without mixer glass beads were developed for the separation of proteins and 4-methylumbelliferyl (MU) sugar derivatives using the small-scale cross-axis coil planet centrifuge. 3 RESULTS: Proteins were well separated from each other and the separation was improved at a low flow rate of the mobile phase. On the other hand, 4-MU sugar derivatives were sufficiently resolved with short separation time at a highflow rate of the mobile phase under satisfactory stationary phase retention. 4 CONCLUSION: Effective separations were achieved using the locular multilayer coil for proteins with aqueous-aqueous polymer phase systems and for 4-MU sugar derivatives with organic-aqueous two-phase solvent systems by inserting a glass bead into each locule.

Identification of marker compounds for Japanese Pharmacopoeia non-conforming jujube seeds from Myanmar.

Jujube seed is a crude drug defined as the seed of Ziziphus jujuba Miller var. spinosa Hu ex H.F. Chou (Rhamnaceae) in the Japanese Pharmacopoeia (JP). Most of the jujube seed in the Japanese markets is imported from China, with the rest obtained from other Asian countries. Here we confirmed the botanical origins of jujube seeds from both China and Myanmar by a DNA sequencing analysis. We found that the botanical origins of the crude drugs from China and Myanmar were Z. jujuba and Z. mauritiana, respectively. Although the jujube seed from China conforms to the JP, that from Myanmar does not. A method for discriminating jujube seeds from China and Myanmar using a chemical approach is thus desirable, and here we sought to identify a compound specific to Z. jujuba. Jujuboside A (1) was identified as a compound specific to Z. jujuba. To establish a purity test of Jujube Seed in the JP against Z. mauritiana, we fractionated the extract of Z. mauritiana seeds and identified frangufoline (2) and oleanolic acid (4) as the marker compounds specific to Z. mauritiana. Thin-layer chromatography (TLC) and gas chromatography-mass spectrometry analyses revealed that the latter compound was useful for testing by TLC analysis. The established TLC conditions were as follows: chromatographic support, silica gel; developing solvent, n-hexane:EtOAc:HCOOH = 10:5:1; developing length, 7 cm; visualization, diluted sulfuric acid; R f value, 0.43 (oleanolic acid).

Isoheleproline: a new amino acid-sesquiterpene adduct from Inula helenium.

A new amino acid-sesquiterpene adduct, isoheleproline (1), was isolated from the roots of Inula helenium (elecampane), together with four known sesquiterpene lactones (2-5). The planar configuration of 1 was elucidated on the basis of spectroscopic data analysis, and the relative configuration of 1 was determined by performing a detailed analysis of NOESY correlations and comparing its physicochemical data with the D- and L-proline adducts of 2 obtained by Michael addition. This is the first report of a new amino acid-sesquiterpene adduct from Inula plants.

Vasorelaxant activity of indole alkaloids from Tabernaemontana dichotoma.

The aim of this study was to search for bioactive natural products from medicinal plants targeting vasorelaxant activity and we found the methanol extract from bark of Tabernaemontana dichotoma showed vasorelaxant activity on rat aorta. We isolated eight indole alkaloids including 10-methoxyalstonerine (1), a new macroline type indole alkaloid, from bark of T. dichotoma. These were respectively identified as 10-methoxyaffinisine (2), lochnerine (3), cathafoline (4), (-)-alstonerine (5), 19,20-dehydro-10-methoxytalcarpine (6), alstonisine (7), and alstonal (8) based on spectroscopic analysis. Among them, sarpagine type (2 and 3), akuammiline type (4), and macroline oxindole type (7 and 8) showed potent vasorelaxant activity. Mechanism of action on vasorelaxant activity of 10-methoxyaffinisine (2), cathafoline (4), and alstonisine (7) was clarified. Effects of 10-methoxyaffinisine (2), cathafoline (4), and alstonisine (7) were partially mediated the NO release from endothelial cells. Furthermore, 10-methoxyaffinisine (2) and alstonisine (7) attribute to the inhibitory effect of VDC and ROC, and cathafoline (4) have inhibitory effect on Ca(2+) influx via ROC. In addition, 10-methoxyaffinisine (2) as a major compound from bark of T. dichotoma showed hypotensive effect on normotensive rats in vivo.

Vasorelaxant effect of FR900359 from Ardisia crenata on rat aortic artery.

A cyclic depsipeptide, FR900359, isolated from Ardisia crenata was evaluated for vasorelaxant effects on rat aortic arteries. FR900359 caused concentration-dependent relaxation (1 nM-10 µM) in phenylephrine-precontracted endothelium-intact aortic rings, which was inhibited by addition of L-NMMA, a NOS inhibitor. In endothelium-denuded rings, the relaxant effect of low concentrations of FR900359 was diminished, but remained at high concentrations. In endothelium-denuded rings, FR900359 at 0.1 µM significantly attenuated high-K(+)-induced contractions and completely inhibited Ca(2+)-induced contractions. These results suggest that the vasorelaxant effect of FR900359 is mediated through the increased release of NO from endothelial cells at low concentrations, and can be attributed to inhibitory effects on voltage-dependent Ca(2+) channel- and receptor-operated Ca(2+) channel-dependent Ca(2+) influx at high concentrations.

Vasorelaxant effects on rat aortic artery by two types of indole alkaloids, naucline and cadamine.

Two indole alkaloids, naucline from Nauclea officinalis and cadamine from Ochreinauclea maingayii, were individually evaluated for vasorelaxant effects on phenylephrine-precontracted aortic rings. Naucline induced concentration-dependent relaxation in aortic rings. Respective EC50 values of naucline on aorta rings with/without endothelium did not show a significant difference, indicating that naucline-induced relaxation was independent of the endothelium. In further experiments with various inhibitors, naucline was found to possess inhibitory effects on both voltage-dependent Ca(2+) channel (VDC)- and receptor-operated Ca(2+) channel (ROC)-dependent Ca(2+) influx in smooth muscle. Cadamine showed concentration-dependent relaxation in endothelium-intact aortic rings, which was inhibited by addition of L-NMMA, NOS inhibitor. These results suggested that the vasorelaxant effect of cadamine is partly mediated through the increased release of NO from endothelial cells. In addition to NO involvement, vasorelaxation induced by cadamine was also attributed to inhibition of both VDC- and ROC-dependent Ca(2+) influx.

Alstiphyllanines I-O, ajmaline type alkaloids from Alstonia macrophylla showing vasorelaxant activity.

Seven new ajmaline type alkaloids, alstiphyllanines I-O (1-7) were isolated from the leaves of Alstonia macrophylla together with six related alkaloids (8-13). Structures and stereochemistry of 1-7 were fully elucidated and characterized by 2D NMR analysis. A series of alstiphyllanines I-O (1-7) as well as the known ajmaline type alkaloids (8-13) showed that they relaxed phenylephrine (PE)-induced contractions against rat aortic ring. Among them, vincamedine (10) showed potent vasorelaxant activity, which may be mediated through inhibition of Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCs) and/or receptor-operated Ca(2+) channels (ROCs) as well as partially mediated the NO release from endothelial cells. The presence of substituents at both N-1 and C-17 may be important to show vasorelaxation activity.

Naucline, a new indole alkaloid from the bark of Nauclea officinalis.

A new indole alkaloid, naucline (1) together with four known alkaloids, angustine (2), angustidine (3), nauclefine (4) and naucletine (5), were isolated from the bark of Nauclea officinalis. The structures of all isolated compounds were elucidated with various spectroscopic methods such as 1D- and 2D- NMR, IR, UV and LCMS-IT-TOF. In addition to that of alkaloid 1, the complete 13C-NMR data of naucletine (5) were also reported. Naucline (1) showed a moderate vasorelaxant activity (90% relaxation at 1 × 10(-5) M) whereas, angustine (2), nauclefine (4), and naucletine (5) showed potent vasorelaxant activity (more than 90% relaxation at 1 × 10(-5) M) on an isolated rat aorta.

Vasorelaxant effect of isoquinoline derivatives from two species of Popowia perakensis and Phaeanthus crassipetalus on rat aortic artery.

Five bisbenzyl isoquinolines (1-5), three benzyl isoquinolines (6-8), four isoquinoline alkaloids (9-12), and two unclassified compounds (13 and 14) from Popowia perakensis and Phaeanthus crassipetalus were evaluated for their vasorelaxant effect on rat aortic arteries. In aortic rings pre-contracted with phenylephrine (PE, 0.3 µM), some of the bisbenzyl isoquinoline alkaloids, benzyl isoquinoline alkaloids, and isoquinoline alkaloids showed clearly vasorelaxant effects at 30 µM. The action of (-)-limacine (4) was deduced to be mediated through the increased release of NO from endothelial cells, and that of pecrassipine A (7) and backebergine (12) partly mediated by NO release. Further, the action of pecrassipine A (7) and backebergine (12) may be attributed to their inhibition of the voltage-dependent Ca(2+) channel and receptor-operated Ca(2+) channel.

Vasorelaxant effects of macrocyclic bis(bibenzyls) from liverworts.

Vasorelaxant effects of a series of bis(bibenzyls) from liverworts such as Marchantia polymorpha and Marchantia paleacea on rat aorta demonstrated that they relaxed phenylephrine (PE)-induced contractions, which may be mediated through the increased release of NO from endothelial cells as well as opening of K(+) channels, and inhibition of Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCs) and/or receptor-operated Ca(2+) channels (ROCs). Structure-activity relationship based on their structures was discussed. The presence of two aromatic rings which can be connected through two atoms bridge spacer may play an important role for vasorelaxant effect.

New vasorelaxant indole alkaloids, villocarines A-D from Uncaria villosa.

Villocarines A-D (1-4), four new indole alkaloids have been isolated from the leaves of Uncaria villosa (Rubiaceae) and their structures were elucidated by 2D NMR methods and chemical correlations. Villocarine A (1) showed vasorelaxation activity against rat aortic ring and showed inhibition effect on vasocontraction of depolarized aorta with high concentration potassium, and also inhibition effect on phenylephrine (PE)-induced contraction in the presence of nicardipine in a Ca(2+) concentration-dependent manner. The vasorelaxant effect by 1 might be attributed mainly to inhibition of calcium influx from extracellular space through voltage-dependent calcium channels (VDC) and/or receptor-operated Ca(2+)-channels (ROC), and also partly mediated through the increased release of NO from endothelial cells and opening of voltage-gated K(+)-channels.

Lancifoliaine, a new bisbenzylisoquinoline from the bark of Litsea lancifolia.

A new bisbenzylisoquinoline, lancifoliaine (1), together with seven known alkaloids--N-allyllaurolitsine (2), reticuline (3), actinodaphnine, norboldine, pallidine, cassythicine and boldine--were isolated from the stem bark of Litsea lancifolia (Lauraceae). In addition to that of lancifoliaine, complete ¹³C-NMR data of N-allyl-laurolitsine (2) was also reported. The alkaloidal structures were elucidated by means of high field 1D- and 2D-NMR IR, UV, and LCMS-IT-TOF spectral data. N-Allyllaurolitsine (2) showed a moderate vasorelaxant activity on isolated rat aorta.