Silica Gel TLC Plates Research Articles

Parameters Characterizing Chromatographic Spots and Separation Effects Determined by Visual and Densitometric Methods

Phloroglucinol (P), niclosamide (N), salicylanilide (S) and thymol (T) were chromatographed on silica gel TLC plates with n‐hexane‐acetone in different volume proportions as mobile phases. Comparison and characterization of chromatographic spots of examined compounds on the basis of resolution (R S ), separation factor (α), constant of the pair separation (R α F ), and ΔR F values were discussed. The R S parameter, serving to evaluate the substance separation, was determined by visual and densitometric methods. It was proven that the R S parameter determined by the visual method for two adjacent substances is always larger than that determined by the densitometric method. It was stated that the densitometric method is correct, objective, and assures standard conditions for the parameter R S determination.

Energy Depletion Inhibits Phosphatidylinositol 3-Kinase/Akt Signaling and Induces Apoptosis via AMP-activated Protein Kinase-dependent Phosphorylation of IRS-1 at Ser-794

Energy depletion activates AMP-activated protein kinase (AMPK) and inhibits cell growth via TSC2-dependent suppression of mTORC1 signaling. Long term energy depletion also induces apoptosis by mechanisms that are not well understood to date. Here we show that AMPK, activated by energy depletion, inhibited cell survival by binding to and phosphorylating IRS-1 at Ser-794. Phosphorylation of IRS-1 at this site inhibited phosphatidylinositol 3-kinase/Akt signaling, suppressed the mitochondrial membrane potential, and promoted apoptosis. Of the treatments promoting energy depletion, glucose deprivation, hypoxia, and inhibition of ATP synthesis in the mitochondria stimulated phosphorylation of IRS-1 at Ser-794 via an LKB1/AMPK-dependent manner, whereas oxidative stress and 2-deoxyglucose stimulated phosphorylation at this site via a Ca2+/calmodulin-dependent protein kinase kinase beta/AMPK axis. These data define a novel pathway that cooperates with other adaptive mechanisms to formulate the cellular response to energy depletion.

Use of SPE-TLC for quality control ofRhodiola roseaextracts

Summary SPE and TLC have been used for qualitative and quantitative analysis of salidroside, rosavin, rosarin, and rosin in commercially available dry extracts from Rhodiola rosea roots. The best separation of all the compounds was achieved on silica gel TLC plates with ethyl acetate–methanol–water, 77 + 13 + 10 (v/v), as mobile phase. UV detection was performed at λ = 215 nm for salidroside and at λ = 245 nm for the rosavins (rosavin, rosarin, and rosin). Detection limits for salidroside and the rosavins were 90 ng and 60 ng per spot, respectively. Results from quantitative analysis confirmed the manufacturer’s declaration of the amounts of salidroside and the rosavins in the extracts.

Densitometric Determination of Fenbendazole in Veterinarian Suspension: Validation of the Method

A simple and rapid densitometric method has been developed for determination of fenbendazole in veterinarian suspension. After extracting the samples with 0.1 N HCL in methanol, the solutions were spotted on precoated silica gel TLC plates, which were eluted with a mixture of dichloromethane‐ethyl acetate–formic acid‐methanol (12:1.0∶0.6∶0.6, v/v). Quantitative evaluation was performed by measuring the absorbance reflectance of the fenbendazole spots at λ=293 nm. The TLC densitometric method is cheap, selective, precise, and accurate, and can be used for routine analysis of suspension in veterinary, pharmaceutical industry quality control laboratories.

Critical Assessment of Various Techniques for the Extraction of Carotenoids and Co-enzyme Q10from the Thraustochytrid Strain ONC-T18

A variety of techniques for extracting carotenoids from the marine Thraustochytrium sp. ONC-T18 was compared. Specifically, the organic solvents acetone, ethyl acetate, and petroleum ether were tested, along with direct and indirect ultrasonic assisted extraction (probe vs bath) methods. Techniques that used petroleum ether/acetone/water (15:75:10, v/v/v) with 3 h of agitation, or 5 min in an ultrasonic bath, produced the highest extraction yields of total carotenoids (29-30.5 microg g-1). Concentrations up to 11.5 microg g-1 of canthaxanthin and 17.5 microg g-1 of beta;-carotene were detected in extracts stored for 6 weeks. Astaxanthin and echinenone were also detected as minor compounds. Extracts with and without antioxidants showed similar carotenoid concentration profiles. However, total carotenoid concentrations were approximately 8% higher when antioxidants were used. Finally, an easy-to-perform and inexpensive method to detect co-enzymes in ONC-T18 was also developed using silica gel TLC plates. Five percent methanol in toluene as a mobile phase consistently eluted co-enzyme Q10 standards and could separate the co-enzyme fractions present in ONC-T18.

Densitometric Determination of Tadalafil Citrate in Tablets: Validation of the Method

A simple and rapid densitometric method has been developed for determination of tadalafil citrate in tablets and its dissolution media. After extracting the samples with acetone, the solutions were spotted onto pre‐coated silica gel TLC plates which were eluted with a mixture of n‐hexane‐ethyl acetate‐methanol (8.0:6.0:2.0, v/v). quantitative evaluation was performed by measuring the absorbance reflectance of the tadalafil citrate spots at λ=285 nm. The TLC‐densitometric method is cheap, selective, precise, and accurate and can be used for routine analysis of tablets in pharmaceutical industry quality control laboratories.

The retention behavior of selected porphyrins on silica gel, polyamide, and cellulose TLC plates

The chromatographic behavior of uroporphyrin I, uroporphyrin III, coproporphyrin I, coproporphyrin III, and protoporphyrin IX has been examined using silica gel 60F 254 , polyamide 11F 254 , and cellulose F TLC plates as stationary phases. Homologous series of n -alcohols, acetonitrile, and tetrahydrofuran were used as mobile phases. Chromatographic retention data and a possible retention mechanism are discussed. The electrostatic interactions operating in these chromatographic systems were analyzed by studying relationship between log (1/ R F ) values and the molecular polarizabilities of the solvents.

DPM1, the Catalytic Subunit of Dolichol-phosphate Mannose Synthase, Is Tethered to and Stabilized on the Endoplasmic Reticulum Membrane by DPM3

Dolichol-phosphate mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-mannose, and C-mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.

Analysis of estrogenic compounds in Polygonum cuspidatum by bioassay and high performance liquid chromatography

The estrogenic activity of traditional Chinese herb– Polygonum cuspidatum Sieb. et Zucc. was investigated by a recombinant yeast screening (YES) assay. Anthraquinones are the main components in the plant, of which emodin is the most abundant one. The ethyl acetate fraction of the ethanol extract of Polygonum cuspidatum was separated on a silica gel TLC plate and seven sub-fractions were collected. The results of bioassay demonstrated that Hzs1 and Hzs6 showed higher estrogenic activities than that of others and the potency of these two compounds were approximately 10 −4 g/L and 10 −3 g/L, respectively. HPLC analysis was performed to determine the activities and the active components. Combining the results of HPLC analysis and estrogenic activity test by YES led to the conclusion that an unknown bioactive compound might exist in the extraction of Polygonum cuspidatum.

Densitometric Determination of Tranexamic Acid in Tablets: Validation of the Method

A simple and rapid densitometric method has been developed for determination of tranexamic acid in tablets and its dissolution media. After extracting the samples with a mixture of a 96% ethanol and water (1 : 1, v/v), the solutions were spotted on precoated silica gel TLC plates, which were eluted with a mixture of n‐butanol–glacial acetic acid–water (8.0 : 2.0 : 2.0, v/v). Quantitative evaluation was performed by measuring the absorbance reflectance of the tranexamic acid spots at λ=488 nm by using ninhydrin reagent. The TLC‐densitometric method is selective, precise, and accurate, and can be used for routine analysis of tablets in the pharmaceutical industry quality control laboratories.

Regulation of Actin Ring Formation by Rho GTPases in Osteoclasts

Actin ring formation is a prerequisite for osteoclast bone resorption. Although gelsolin null osteoclasts failed to exhibit podosomes, actin ring was observed in these osteoclasts. Wiscott-Aldrich syndrome protein (WASP) was observed in the actin ring of gelsolin null osteoclast. Osteoclasts stimulated with osteopontin simulated the effects of Rho and Cdc42 in phosphatidylinositol 4,5-bisphosphate (PIP2) association with WASP as well as formation of podosomes, peripheral microfilopodia-like structures, and actin ring. To explore the potential functions of Rho and Cdc42, TAT-mediated delivery of Rho proteins into osteoclasts was performed. Although Rho and Cdc42 are required for actin ring formation, transduction of either one of the proteins alone is insufficient for this process. Addition of osteopontin to osteoclasts transduced with Cdc42Val12 or transduction of osteoclasts with both RhoVal14 and Cdc42Val12 augments the formation of WASP-Arp2/3 complex and actin ring. Neomycin, an antibiotic, blocked the effects of osteopontin or TAT-RhoVal14 on PIP2 interaction with WASP. WASP distribution was found to be cytosolic in these osteoclasts. Depletion of WASP by short interfering RNA-mediated gene silencing blocked actin polymerization as well as actin ring formation in osteoclasts. These results suggest that Rho-mediated PIP2 interaction with WASP may contribute to the activation and membrane targeting of WASP. Subsequent interaction of Cdc42 and Arp2/3 with WASP may enhance cortical actin polymerization in the process of actin ring formation in osteoclasts.

Inducible Nitric-oxide Synthase and NO Donor Induce Insulin Receptor Substrate-1 Degradation in Skeletal Muscle Cells

Chronic inflammation plays an important role in insulin resistance. Inducible nitric-oxide synthase (iNOS), a mediator of inflammation, has been implicated in many human diseases including insulin resistance. However, the molecular mechanisms by which iNOS mediates insulin resistance remain largely unknown. Here we demonstrate that exposure to NO donor or iNOS transfection reduced insulin receptor substrate (IRS)-1 protein expression without altering the mRNA level in cultured skeletal muscle cells. NO donor increased IRS-1 ubiquitination, and proteasome inhibitors blocked NO donor-induced reduction in IRS-1 expression in cultured skeletal muscle cells. The effect of NO donor on IRS-1 expression was cGMP-independent and accentuated by concomitant oxidative stress, suggesting an involvement of nitrosative stress. Inhibitors for phosphatidylinositol-3 kinase, mammalian target of rapamycin, and c-Jun amino-terminal kinase failed to block NO donor-induced IRS-1 reduction, whereas these inhibitors prevented insulin-stimulated IRS-1 decrease. Moreover iNOS expression was increased in skeletal muscle of diabetic (ob/ob) mice compared with lean wild-type mice. iNOS gene disruption or treatment with iNOS inhibitor ameliorated depressed IRS-1 expression in skeletal muscle of diabetic (ob/ob) mice. These findings indicate that iNOS reduces IRS-1 expression in skeletal muscle via proteasome-mediated degradation and thereby may contribute to obesity-related insulin resistance.

Densitometric determination of desloratadine in tablets, and validation of the method

Summary A simple and rapid densitometric method has been developed for determination of desloratadine in tablets. After extraction of the samples with 96% ethanol the solutions were spotted on precoated silica gel TLC plates and these were eluted with ethyl acetate– n-butanol–25% aqueous ammonia–methanol, 21 + 5.0 + 4.0 + 5.0 (v/v), as mobile phase. Quantitative evaluation was performed by measuring the absorbance reflectance of the desloratadine spots at λ = 279 nm. The TLC–densitometric method is selective, precise, and accurate and can be used for routine analysis of tablets in pharmaceutical industry quality control laboratories.

Densitometric Determination of Allylestrenol in Tablets, and Validation of the Method

A simple and rapid densitometric method has been developed for determination of allylestrenol in tablets. After extracting the samples with 96% ethanol, the solutions were spotted on pre‐coated silica gel TLC plates, which were eluted with a mixture of n‐hexane–ethyl acetate–dichloromethane (9.0:2.0:0.2, v/v). Quantitative evaluation was performed by measuring the absorbance reflectance of the allylestrenol spots at λ = 609 nm, after reaction with anisaldehyde–sulphuric acid reagent (5 min, 100°C). The TLC‐densitometry method is selective, precise, and accurate, and can be used for routine analysis of tablets in pharmaceutical industry quality control laboratories.

Emission Quenching of a Poly(Phenylene Ethynylene) (PPE) in the Solid State

ABSTRACTThe ability of a poly(phenylene ethynylene) conjugated polyelectrolyte to detect a small molecule quencher in the solid state was assessed by coating the polymer onto a plastic-backed, non-fluorescent, silica-gel TLC plate. The efficiency of the emission quenching process was quantified by noting the resulting loss of emission with increasing quencher concentration, in accordance with the Stern-Volmer relation. The emission quenching induced by the addition of the analyte occurred with Stern-Volmer constants in the range of 103 M−1.

Screening of antifungal agents using ethanol precipitation and bioautography of medicinal and food plants

In the search for bioactive compounds, bioautography and ethanol precipitation of macromolecules (proteins, polysaccharides, etc.) of plant aqueous extracts were associated in an antifungal screening. Thus, the supernatants, precipitates (obtained by ethanol precipitation) and aqueous extracts were investigated of medicinal and fruit bearing plants used against skin diseases by the Brazilian population. The agar diffusion and broth dilution methods were used to assess the activity against three fungi: Candida albicans, Trichophyton rubrum and Cryptococcus neoformans. The results, evaluated by the diameter of the inhibition zone of fungal growth, indicate that six plant species, among the 16 investigated, showed significant antifungal activity. The minimal inhibitory concentration (MIC) was determined on plant extracts that showed high efficacy against the tested microorganisms. The most susceptible yeast was Trichophyton rubrum and the best antifungal activity was shown by Xanthosoma sagittifolium supernatant. The bioautography was performed only for the aqueous extracts and supernatants of those plants that showed antifungal activity against Candida albicans and Cryptococcus neoformans, using n-butanol/acetic acid/water (BAW) 8:1:1 to develop silica gel TLC plates. Clear inhibition zones were observed for aqueous extracts of Schinus molle (R f 0.89) and Schinus terebinthifolius (R f 0.80) against Candida albicans, as for supernatant of Anacardium occidentale (R f 0.31) against Cryptococcus neoformans. The separation of macromolecules from metabolites, as in the case of Anacardium occidentale, Solanum sp. and Xanthosoma sagittifolium, enhances antifungal activity. In other cases, the antifungal activity is destroyed, as observed for Momordica charantia, Schinus molle and Schinus terebinthifolius.

Variable recoveries of fatty acids following the separation of lipids on commercial silica gel TLC plates: Selective loss of unsaturated fatty acids on certain brands of plates

Since we recently noticed poor recoveries of unsaturated fatty acids (UFA) when the parent lipids were first separated on TLC plates, we investigated the source of this error by examining several variables, including the brand of TLC plate, nature of the lipid, and conditions of methylation. Of the five commercial brands of plates used, two (Baker and Whatman) showed loss of UFA, and three (Alltech Hardlayer, Alltech Softlayer, and Merck) did not. This loss occurred in both neutral and phospholipids, did not affect saturated acids, and was independent of the methylation reagent used. No loss occurred, however, if the lipids were eluted from the silica gel before methylation, indicating that the loss is due to oxidation of UFA in presence of certain brands of silica gel. These results show that some brands of TLC plates may be unsuitable for lipid analysis, if the aim is to determine the fatty acid composition by GC using direct methylation.

Ethylene Production and Endogenous Gibberellin Content in Chrysanthemum Plants as Affected by Uniconazole, Brushing and Aminoethoxyvinylglycine Treatments

Chrysanthemum (Dendranthema grandiflora (Ramat.) Kitam.) plants, sprayed with a 25 ppm uniconazole one month after planting, evolved ethylene immediately and attained the highest yield on the third day. Plants that were brushed (mechanical stress, MS) for 30 days produced more ethylene on the third day than did uniconazole-sprayed plants and maintained their high production. Plants treated with aminoethoxyvinylglycine (AVG) yielded the lowest levels of ethylene and displayed no peak. Application of AVG to uniconazole-sprayed or brushed plants decreased ethylene evolution. Gibberellic acid (GA)-like substances in the control plants separated into 4 bands on silica gel TLC plates; authentic GA1 GA3, and GA7 were detected at the same Rf in the control plants. GA1- and GA3-like substance contents decreased markedly in plants sprayed with uniconazole or brushed. The application of AVG alone did not affect GA- like substance content, nor did it enhance GA activities in uniconazole-sprayed plants. Thus, growth of chrysanthemum plants is controlled not only by GA activities but also by ethylene.

Densitometric determination of betamethasone dipropionate and salicylic acid in lotions, and validation of the method

A simple and rapid densitometric method has been developed for determination of betamethasone dipropionate and salicylic acid in lotions. The samples were diluted with 96% ethanol and spotted on precoated silica gel TLC plates which were then eluted with ethanol (96%)-toluene-chloroform-glacial acetic acid, 6.0 + 20 + 14 + 0.5 ( v/v ). Quantitative evaluation was performed by measuring the absorbance reflectance of the betamethasone dipropionate and salicylic acid spots at λ = 250 and 310 nm, respectively. This densitometric TLC method is selective, precise, and accurate and can be used for routine analysis of lotions in pharmaceutical industry quality-control laboratories.

Slit protein-mediated inhibition of CXCR4-induced chemotactic and chemoinvasive signaling pathways in breast cancer cells.

Slit, which mediates its function by binding to the Roundabout (Robo) receptor, has been shown to regulate neuronal and CXCR4-mediated leukocyte migration. Slit-2 was shown to be frequently inactivated in lung and breast cancers because of hypermethylation of its promoter region. Furthermore, the CXCR4/CXCL12 axis has been reported recently to be actively involved in breast cancer metastasis to target organs such as lymph nodes, lung, and bone. In this study, we sought to characterize the effect of Slit (=Slit-2) on the CXCL12/CXCR4-mediated metastatic properties of breast cancer cells. We demonstrate here that breast cancer cells and tissues derived from breast cancer patients express Robo 1 and 2 receptors. We also show that Slit treatment inhibits CXCL12/CXCR4-induced breast cancer cell chemotaxis, chemoinvasion, and adhesion, the fundamental components that promote metastasis. Slit had no significant effect on the CXCL12-induced internalization process of CXCR4. In addition, characterization of signaling events revealed that Slit inhibits CXCL12-induced tyrosine phosphorylation of focal adhesion components such as RAFTK/Pyk2 at residues 580 and 881, focal adhesion kinase at residue 576, and paxillin. We found that Slit also inhibits CXCL12-induced phosphatidylinositol 3-kinase, p44/42 MAP kinase, and metalloproteinase 2 and 9 activities. However, it showed no effect on JNK and p38 MAP kinase activities. To our knowledge, this is the first report to analyze in detail the effect of Slit on breast cancer cell motility as well as its effect on the critical components of the cancer cell chemotactic machinery. Studies of the Slit-Robo complex may foster new anti-chemotactic approaches to block cancer cell metastasis.