Silica Gel Thin-layer Plates Research Articles

Effect of superfine pulverization technology on the determination of vitamin C content in Fructus rosae laxae

Superfine pulverization and ordinary pulverization were used to obtain powders with different particle sizes in different parts of Fructus rosae laxae. High-performance thin-layer chromatography (HPTLC) was used to carry out a simple, fast, qualitative, and quantitative analysis of vitamin C in Fructus rosae laxae. The effects of superfine pulverization and ordinary pulverization on the vitamin C content in the whole fruit, pulp, and seed of Fructus rosae laxae were compared. A G F254 silica gel thin-layer plate was utilized, and ethyl acetate, anhydrous ethanol, and water in (10:6:3.75, v/v) ratio were used as a developer. The scanning wavelength was 273 nm. Results showed that the Rf value of vitamin C was 0.61, the linear range was 0.25–1.2 μg, and the correlation coefficient was 0.9959, indicating good linear correlation. Precision RSD was < 2%, indicating good instrument precision. The sample was stable within 40 min at room temperature, and the RSD was < 3%. The average recovery of vitamin C was 98.39%, and the established method was reliable. The content of vitamin C in the whole fruit, pulp, and seed of Fructus rosae laxae after superfine pulverization was higher than that obtained from ordinary pulverization. The content of vitamin C in the pulp was the highest after superfine pulverization.

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.

A novel “target constituent knock-out” strategy coupled with TLC, UPLC–ELSD and microcalorimetry for preliminary screening of antibacterial constituents in Calculus bovis

A novel “target constituent knock-out” strategy was proposed and applied for preliminary screening of antibacterial constituents in Calculus bovis ( C. bovis). This strategy was accomplished through the following steps: (1) the single constituents (A–F) in C. bovis samples were knocked out on the Silica Gel thin-layer plates by thin-layer chromatography (TLC); (2) these knocked-out constituents were identified by ultra performance liquid chromatography–evaporative light scattering detection (UPLC–ELSD); (3) the antibacterial activities of these knocked-out constituents and C. bovis samples on Staphylococcus aureus ( S. aureus) were evaluated by microcalorimetry combined with principal component analysis (PCA); (4) the activities of these knocked-out constituents and the total extract of C. bovis, also the interaction properties between these single constituents and the total extract were elucidated. The results showed that the sum of inhibitory ratio ( I) of constituents A–F (202.0%) was 5-fold of the I of C. bovis sample (38.01%), showing that these knocked-out constituents had strong antagonistic effects on each other in C. bovis sample and the antagonistic extent was 81.18%. And we found that the key antibacterial composition of C. bovis was not a single component, also not the high content component (cholic acid, CA), but constituent F, which was the combinatorial composition of deoxycholic acid (DCA) and hyodeoxycholic acid (HDCA). Constituent F revealed over 33-fold high activity of the sum of DCA and HDCA activity in solo-use, showing strong synergistic effect between DCA and HDCA. In addition, constituents A–E had significant antagonistic effects on constituent F. Our study indicates that this proposed “target constituent knock-out” strategy is a useful approach for screening active constituents and elucidating the multi-component interactions in C. bovis, further providing some reference for understanding the pharmacodynamic actions, controlling the quality of Chinese materia medicas (CMMs) and discovering new drugs.

Inhibition of Mammalian Target of Rapamycin Signaling by 2-(Morpholin-1-yl)pyrimido[2,1-α]isoquinolin-4-one

Signaling through the mammalian target of rapamycin (mTOR) is hyperactivated in many human tumors, including hamartomas associated with tuberous sclerosis complex (TSC). Several small molecules such as LY294002 inhibit mTOR kinase activity, but they also inhibit phosphatidylinositol 3-kinase (PI3K) at similar concentrations. Compound 401 is a synthetic inhibitor of DNA-dependent protein kinase (DNA-PK) that also targets mTOR but not PI3K in vitro (Griffin, R. J., Fontana, G., Golding, B. T., Guiard, S., Hardcastle, I. R., Leahy, J. J., Martin, N., Richardson, C., Rigoreau, L., Stockley, M., and Smith, G. C. (2005) J. Med. Chem. 48, 569-585). We used 401 to test the cellular effect of mTOR inhibition without the complicating side effects on PI3K. Treatment of cells with 401 blocked the phosphorylation of sites modified by mTOR-Raptor and mTOR-Rictor complexes (ribosomal protein S6 kinase 1 Thr(389) and Akt Ser(473), respectively). By contrast, there was no direct inhibition of Akt Thr(308) phosphorylation, which is dependent on PI3K. Similar effects were also observed in cells that lack DNA-PK. The proliferation of TSC1-/- fibroblasts was inhibited in the presence of 401, but TSC1+/+ cells were resistant. In contrast to rapamycin, long-term treatment of TSC1-/- cells with 401 did not up-regulate phospho-Akt Ser(473). Because increased Akt activity promotes survival, this may explain why the level of apoptosis was increased in the presence of 401 but not rapamycin. These results suggest that mTOR kinase inhibitors might be more effective than rapamycins in controlling the growth of TSC hamartomas and other tumors that depend on elevated mTOR activity.

Key Role for Ceramides in Mediating Insulin Resistance in Human Muscle Cells

Elevated non-esterified fatty acids, triglyceride, diacylglycerol, and ceramide have all been associated with insulin resistance in muscle. We set out to investigate the role of intramyocellular lipid metabolites in the induction of insulin resistance in human primary myoblast cultures. Muscle cells were subjected to adenovirus-mediated expression of perilipin or incubated with fatty acids for 18 h, prior to insulin stimulation and measurement of lipid metabolites and rates of glycogen synthesis. Adenovirus-driven perilipin expression lead to significant accumulation of triacylglycerol in myoblasts, without any detectable effect on insulin sensitivity, as judged by the ability of insulin to stimulate glycogen synthesis. Similarly, incubation of cells with the monounsaturated fatty acid oleate resulted in triacylglycerol accumulation without inhibiting insulin action. By contrast, the saturated fatty acid palmitate induced insulin resistance. Palmitate treatment caused less accumulation of triacylglycerol than did oleate but also induced significant accumulation of both diacylglycerol and ceramide. Insulin resistance was also caused by cell-permeable analogues of ceramide, and palmitate-induced resistance was blocked in the presence of inhibitors of de novo ceramide synthesis. Oleate co-incubation completely prevented the insulin resistance induced by palmitate. Our data are consistent with ceramide being the agent responsible for insulin resistance caused by palmitate exposure. Furthermore, the triacylglycerol derived from oleate was able to exert a protective role in sequestering palmitate, thus preventing its conversion to ceramide.

Regulation of intestinal NPC1L1 expression by dietary fish oil and docosahexaenoic acid

To address the effect of the n-3 fatty acid, docosahexaenoic acid (22:6), on proteins that play a role in cholesterol absorption, CaCo-2 cells were incubated with taurocholate micelles alone or micelles containing 22:6 or oleic acid (18:1). Compared with controls or 18:1, 22:6 did not interfere with the cellular uptake of micellar cholesterol. Apical cholesterol efflux was enhanced in cells incubated with 22:6. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was decreased by 22:6. 22:6 decreased Niemann-Pick C1-Like 1 (NPC1L1) protein and mRNA levels without altering gene or protein expression of ACAT2, annexin-2, caveolin-1, or ABCG8. Peroxisome proliferator-activated receptor delta (PPARdelta) activation decreased NPC1L1 mRNA levels and cholesterol trafficking to the endoplasmic reticulum, suggesting that 22:6 may act through PPARdelta. Compared with hamsters fed a control diet or olive oil (enriched 18:1), NPC1L1 mRNA levels were decreased in duodenum and jejunum of hamsters ingesting fish oil (enriched 22:6). In an intestinal cell, independent of changes in ABCG8 expression, 22:6 increases the apical efflux of cholesterol. 22:6 interferes with cholesterol trafficking to the endoplasmic reticulum by the suppression of NPC1L1, perhaps through the activation of PPARdelta. Moreover, a diet enriched in n-3 fatty acids decreases the gene expression of NPC1L1 in duodenum and jejunum of hamster.

Novel Tumor Necrosis Factor-responsive Mammalian Neutral Sphingomyelinase-3 Is a C-tail-anchored Protein

Two genes encoding neutral sphingomyelinases-1 and -2 (sphingomyelin phosphodiesterases-2 and -3) have been recently identified that hydrolyze sphingomyelin to phosphorylcholine and ceramide. Data bank searches using a peptide sequence derived from a previously purified bovine neutral sphingomyelinase (nSMase) allowed us to identify a cDNA encoding a novel human sphingomyelinase, nSMase3, that shows only a little homology to nSMase1 and -2. nSMase3 was biochemically characterized by overexpression in a yeast strain, JK9-3ddeltaIsc1p, lacking endogenous SMase activity. Similar to nSMase2, nSMase3 is Mg2+-dependent and shows optimal activity at pH 7, which is enhanced in the presence of phosphatidylserine and inhibited by scyphostatin. nSMase3 is ubiquitously expressed as a 4.6-kb mRNA species. nSMase3 lacks an N-terminal signal peptide, yet contains a 23-amino-acid transmembrane domain close to the C terminus, which is indicative for the family of C-tail-anchored integral membrane proteins. Cellular localization studies with hemagglutinin-tagged nSMase3 demonstrated colocalization with markers of the endoplasmic reticulum as well as with Golgi markers. Tumor necrosis factor stimulates rapid activation of nSMase3 in MCF7 cells with peak activity at 1.5 min, which was impaired by expression of dominant negative FAN.

Thin-Layer Chromatography Analysis of Human CYP3A-Catalyzed Testosterone 6β-Hydroxylation.

Testosterone and other steroid hormones have been studied as prototypic examples of endogenous substrates for hepatic cytochrome P450 (P450) enzymes. CYP3A enzymes from various species, including human, metabolize testosterone by a 6β-hydroxylation reaction, which is unique to this P450 subfamily. A thin-layer chromatographic method is described for the determination of 6β-hydroxytestosterone formed enzymatically by incubation of [14C]-testosterone with cDNA-expressed CYP3A enzymes or liver microsomes. 14C-labeled enzymatic products are applied to silica gel thin-layer plates, which are developed sequentially with methylene chloride:acetone (80:20) followed by chloroform, ethyl acetate, and absolute ethanol (80:20:14). Metabolite quantification is performed by autoradiography and liquid scintillation counting. This method is applicable to enzymatic studies for the determination of CYP3A-dependent testosterone 6β- hydroxylation activity in both human and animal liver microsomes.

Thin‐layer chromatography separation of enantiomers of verapamil using macrocyclic antibiotic as a chiral selector

Silica gel thin-layer chromatography plates impregnated with macrocyclic antibiotic, vancomycin, as chiral selector were prepared and used for the resolution of (+/-)-verapamil. A mobile phase system of acetonitrile-methanol-water (15:2.5:2.5, v/v) was worked out systematically. The effects of chiral selector, temperature and pH on resolution were also studied. The spots were detected with iodine vapors and the detection limit was found to be 0.074 microg of each enantiomers.

High frame-rate imaging of surface pressure distribution using a porous pressure-sensitive paint

The aim of the present work is to demonstrate the feasibility of a porous pressure-sensitive paint (PSP) for time-resolved surface pressure measurements in unsteady high-speed flows. The porous PSP was composed of bathophenanthroline ruthenium(II) complex, Ru(Ph2-phen) and a silica-gel thin-layer chromatography aluminium plate. The dynamic response of the porous PSP was characterized by a point-wise luminescence intensity measurement conducted in a shock tube facility. The result showed that the time constant of the porous PSP was 13.6 µs. The porous PSP was then applied to the surface pressure distribution imaging of an unsteady flow induced in a two-dimensional Laval nozzle by using a fast-framing complementary metal oxide semiconductor camera. It was clearly shown that the porous PSP well captured the shock-wave motion of the order of kilohertz during the starting process of the supersonic nozzle in a qualitative manner.

B306 感圧塗料を用いた回転物体上における表面圧力分布の可視化

The objective of the present work is to study the feasibility of the pressure-sensitive paint (PSP) technique for pressure measurements on rotating objects. The PSP used was composed of a porphyrin derivative (H 2TFPP) and a silica-gel thin-layer chromatography plate. The illumination sources were arrays of blue LED, and the detecter was a CCD camera with 12 bit intensity resolution. In the experiments, the shutter of the CCD camera was left open and a rotating model was flashed repeatedly as it came into position, building up a phase-averaged image on the CCD camera. The present preliminary experiments show that the deformation of the model under air loads seriously affects the PSP measurements on rotating bodies.

An improved method for extraction and separation of photosynthetic pigments

The method for extracting and separating hydrophobic photosynthetic pigments proposed by Katayama et al. (Japanese Journal of Phycology, 42, 71-77, 1994) has been improved to introduce it to student laboratories at the senior high school level. Silica gel powder was used for removing water from fresh materials prior to extracting pigments by a mixture of organic solvents that was also used for chromatographic separation of the pigments. A small silica gel thin-layer plate or a paper strip was used for separating the pigments. The improved method may be applicable to all kinds of plant materials including algae, is easier than most other methods, and can lead to more successful results in separating these pigments by both thin-layer chromatography and paper chromatography. The method has been carried out in student laboratories in some senior high schools and universities in Japan. The results indicate that this laboratory exercise is effective for students to recognise the unity and diversity of plants. Therefore, this laboratory seems to be useful for teaching plant systematics as well as for teaching photosynthesis.

Quantitative determination of diazepam, nitrazepam and flunitrazepam in tablets using thin-layer chromatography–densitometry technique

A simple and reliable assay for diazepam, nitrazepam and flunitrazepam in tablets by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC)–densitometry is described. A quantity of a ground tablet mass, equal to the average weight of one tablet was sonicated in MeOH, mixed with appropriate internal standard, filtered and either injected directly into the liquid chromatograph, or after evaporation and reconstitution of an aliquot of the extract, was spotted on a silica gel thin-layer plate. A variable UV detector, operated at 254 nm was employed in both procedures. A C18, reversed phase 7 μm column was used for HPLC analysis; the mobile phase was a 1:1 (v/v) mixture of MeOH (40 °C) and 0.01 M phosphate buffer (pH 7, 80 °C). The TLC plate was developed in an unsaturated chromatographic chamber containing 100 ml chloroform–acetone (9:1); at room temperature, the mobile phase was allowed to travel 15 cm. The percentage of the active ingredient content of each tablet obtained by both procedures, was in the range of the stated amount except for one brand of diazepam tablets which contained ∼23% less active ingredient than the minimum prescribed amount. The TLC densitometry, although yields slightly higher values than the HPLC method, is preferred due to its simplicity, ease and low cost.

Optical measurements of high-frequency pressure fluctuations using apressure-sensitive paint and Cassegrain optics

Optical measurements of high-frequency pressure fluctuations have been performedto examine the dynamic response of a fast-responding porous pressure-sensitivepaint (PSP). The PSP was composed of meso-tetra(pentafluorophenyl) porphineand a commercial silica-gel thin-layer chromatography plate. Pressure fluctuationsat frequencies on the order of kilohertz were produced on a flat plate by choppingan underexpanded air-jet and then measured by using a newly developed,optical pressure measurement system (OPMS) with a Cassegrain-typeoptical probe. Pressure traces obtained by the present OPMS and theiramplitude spectra were compared with those obtained by a piezo-resistivepressure transducer. The present results demonstrate the feasibility of thepresent OPMS for measuring high-frequency pressure fluctuations witha high spatial resolution and reveal that the present porous PSP canfollow pressure fluctuations at frequencies of several kilohertz at least.

Formation of (R)-2,3-dihydrogeranylgeranoic acid from geranylgeraniol in rat thymocytes.

In a metabolic study of [1-(14)C]geranylgeranial involving rat thymocytes, the radioactivity was mainly incorporated into two metabolites, Z1 and Z2, the latter moving slower than the former on a silica-gel thin-layer plate. The time course of Z1 and Z2 formation superficially suggested a precursor-product relationship between Z1 and Z2. The two metabolites were chemically converted to their methyl esters on treatment with trimethylsilyl diazomethane. Z1 was cochromatographed with E,E,E-geranylgeranoic acid (GGA). Z2 was prepared in a large quantity from geranylgeranial using thymocytes, and purified by TLC followed by ESI (negative ion mode) or EI mass-spectrometry. The observation of a negative ion at m/z 305 on ESI and a molecular ion at m/z 306 (C(20)H(34)O(2)) with fragments similar to GGA on EI implied that Z2 was dihydroGGA, which has been detected in the urine and serum of patients with Refsum disease. The EI mass spectrum of (R)-2,3-dihydroGGA was identical to that of Z2. The diastereomeric amide synthesized from metabolite Z2 with (R)-1-(1-naphtyl)ethylamine was cochromatographed with (R acid, R) amide, not with (S acid, R) amide, which were similarly synthesized from (R)- and (S)-2,3-dihydroGGAs, respectively. In another metabolic study on [1-(14)C]geranylgeraniol (GGOH), the radioactivity was similarly incorporated into a metabolite corresponding to (R)-2,3-dihydroGGA. (R)-2,3-DihydroGGA induced DNA ladder formation with a maximum at 15 mciroM in thymocytes. However, 2,3-dihydrofarnesoic acid did not induce it at all.

Tetrahymena thermophila disA mutants exhibit aberrant levels of phosphatidylethanolamine

Summary Tetrahymena thermophila wild type strain B 1868 and T. thermophila disA mutant were examined for total phospholipid composition by radiolabeling whole cells with [ 32 P]orthophosphate, [ 3 H]myristic acid, and [ 3 H]acetate. Percent distribution of radioactivity into individual phospholipid classes was determined by scintillation counting or by autoradiography following chromatography of total lipid extracts on silica gel thin layer plates. A significant reduction in radiolabel incorporation into the disA mutant phosphatidylethanolamine fraction was observed in all radiolabeling experiments.

Labeling of Influenza Virus with Alkylamine-Modified Horseradish Peroxidase

A procedure for labeling influenza viruses with alkylamine-modified horseradish peroxidase (HRP) was developed to study virus receptor specificities in a virus overlay assay. Six alkylamine derivatives of HRP were prepared by the periodate oxidation method. Human influenza virus A/PR/8/34 (H1N1) was labeled with the alkylamine derivatives, and a variety of parameters for labeling were tested in a fetuin-immobilized microplate solid-phase assay. The labeling efficiency of influenza virus with the derivatives of HRP was affected by the alkyl chain length of the derivatives. The most effective derivative for labeling the virus being tested was octylamine-modified HRP (octyl-HRP). The labeling profile of A/PR/8/34 by octyl(C8H17)-HRP was analyzed by sucrose density gradient centrifugation. The labeling had no effect on viral hemagglutinating activity and fetuin binding specificity. The specific binding of the HRP-labeled human influenza viruses (A/PR/8/34 and B/Lee/40) to glycolipids was demonstrated by a virus overlay assay. In this virus-HRP labeled system, the virions which bound to gangliosides developed on a silica gel thin-layer plate were detected simply and rapidly.

Functionalization on surfaces of solid materials by alcoholic plasma-polymer films.

Hydrophilic plasma-polymer films were prepared from 2- propyn-1-ol (propargyl alcohol, PA) on surfaces of solid materials. The polymer enhanced its wettability by increasing the amount of water added to the PA monomer. Functional property of the polymer was first evaluated by coating of the plasma-polymerized PA (PPPA) film on a silica gel thinlayer plate. Chromatographic behaviors of some organic compounds was significantly altered by interaction between the polymer and the organic samples. Impregnation of chondroitin sulfate in the silica gel and following immobilization with the plasma-polymer exhibited affinities for Co2+, Fe2+, and Cu2+ depending upon the amount of chondroitin sulfate immobilized. PPPA showed humidity-electroconductive property that was usable as a sensing device of a hygrometer. An immobilized glucose oxidase (GOD) membrane was prepared by impregnating GOD in a porous membrane and coated with PPPA film. The membrane was attached to a dissolved oxygen electrode for composing a glucose sensor and the sensor worked with linear response up to the glucose concentration of 2mM.

Thin-layer chromatographic detection of ivermectin in cattle serum

A study was conducted on the detection of ivermectin (22,23-dihydroavermectin B 1) in cattle serum by thin-layer chromatography (TLC) after derivatization of this parasiticide by the known reaction with trifluoroacetic anhydride—1-methylimidazole and visual examination of the chromatograms under long-wavelength ultraviolet light. By derivatization of reference samples of ivermectin in acetonitrile, approximately 0.1 ng of a highly fluorescent material, tentatively identified as 2,5,6-tetradehydro-5,7-dideoxy-22,23-dihydroavermectin B 1, could be detected on silica-gel thin layer plates. Extraction of fortified serum samples with methyl tert.-butyl ether followed by derivatization, hydrolysis, partitioning between water—hexane and TLC gave a limit of detection of 1–2 ng/ml. With these simple techniques ivermectin could be detected in cattle serum for 3–4 weeks after subcutaneous treatment of Hereford heifers.

Resolution of amino acid racemates on borate-gelled guaran-impregnated silica gel thin-layer chromatographic plates

Borate-gelled guaran-impregnated silica gel TLC plates act as a very good chiral chromatographic media. Tetracoordinated boron is complexed to the cis-OH pairs in guaran polysaccharide, creating a chiral environment around boron. The complexed boron can undergo a ligand-exchange reaction or stereoselective dual H-bonding to a bis-oxygen or N,O-ligand, such as conformationally favourable glycol, α-hydroxy acids and α-amino acids. These reactions, due to chiral selectivity, result in a difference in the migration rates of the enantiomers of a racemic mixture on a TLC plate. This provided a simple, rapid and effective method for the resolution of α-amino acid racemates.