AgNO3-impregnated Silica Gel Research Articles

Identification and Quantification of Germacrene D from the Extracts of Zanthoxylum ovalifolium Leaves by AgNO3-TLC Densitometric Method

Germacrene D an important sesquiterpenoid has been extracted from leaves of Zanthoxylum ovalifoilum and high performance thin-layer chromatographic method has been developed for its simultaneous identification and determination. The compound was extracted by cold extraction and separated and identified on a AgNO3 impregnated silica gel 60 F254 HPTLC plates with dichloromethane:acetone (9.8:0.2 v/v) as mobile phase. The quantification of geremcare D was carried out by densitometry scanning performed at λ = 254 nm by reflectance scanning. This facilitated a well-resolved band for the compound with a Rf value of 0.46 ± 0.01. Linear regression concentration analysis data for mean calibration plots showed good linear relationship in the range of 1000-10000 ng/mL, with a correlation coefficient, slope and intercept of 0.9985 ± 0.0004, 21.62 ± 0.435 and 3308.87 ± 10.24, respectively. The minimum amount of germacrene D that could be authentically detected (LOD) and quantified (LOQ) were determined and found to be 212.07 and 521.23 ng/spot, respectively. The method was validated by considering the parameters linearity, precision, accuracy, specificity and robustness wherein, it is evident from the results that the proposed method was simple, safe and reliable.

Efficient Approach to Medium-sized Cyclic Molecules Containing (E)-Alkene via Z to E Photochemical Isomerization in the Presence of AgNO3-impregnated Silica Gel

Efficient synthesis of medium-sized cyclic molecules containing an (E)-alkene was performed via the highly (E)-selective photochemical isomerization of the (Z)-isomer, facilitated by AgNO3-impregnated silica gel.

Synthesis and Stereochemical Analysis of Planar-Chiral (E)-4-[7]Orthocyclophene.

An efficient synthesis of (E)-4-[7]orthocyclophene (E)-1 via photochemical isomerization of (Z)-1 has been achieved. The key intermediate (Z)-1 was synthesized from commercially available 2-(hydroxymethyl)benzenepropanol (3) in five steps: (i) group-selective Mitsunobu reaction with CH2═CHCH2CH(SO2Ph)2, (ii) oxidation of alcohol, (iii) olefination, (iv) RCM, and (v) removal of sulfones in an overall yield of 73%. The photochemical isomerization of (Z)-1 was efficiently performed in the presence of AgNO3-impregnated silica gel (AgNO3/SiO2). The resulting (E)-1 shows dynamic planar chirality at rt. Enantioenriched (E)-1 was prepared by the HPLC separation of enantiomers using a chiral stationary phase, and the absolute stereochemistry was determined by X-ray diffraction analysis of the Pt-coordinated crystalline derivative. The planar chirality of (E)-1 can be converted into the central chirality of carbon; e.g., the oxidation of (R)-(E)-1 using DMDO provided epoxide (8S,9S)-9 in a stereospecific manner. Furthermore, the Lewis acid-promoted reaction of (8S,9S)-9 afforded a unique tricyclic compound (8S,9S)-10 in an excellent yield and in a stereospecific manner.

4-Me-6E,8E-hexadecadienoic acid isolated from a marine-derived strain of Clonostachys rosea reduces viability of MCF-7 breast cancer cells and gene expression of lipogenic enzymes

Conjugated fatty acids (CFA) have attracted particular attention because of their remarkable biological activities such as anti-carcinogenic, anti-obesity, antithrombotic and anti-atherogenic [1]. The main natural sources of CFA correspond to ruminant milk, algae and seed oils of several plants [2], but fungi have been widely recognized for their production of essential FA such as arachidonic or docosahexaenoic acids which have a major interest in human health and nutrition. Our attention was focused on a marine-derived strain of Clonostachys rosea isolated from marine sediments of the Loire estuary (France), which produced high amounts of triglycerides and an original branched CFA, first identified as 4-Me-6,8-hexadecadienoic acid (4-Me-6,8 – 16:2). In order to optimize the production of this CFA to undergo pharmacological studies, an OSMAC approach has been performed on 7 culture media. Cultures on DCA medium showed the highest production with 19% of total FA. After fungal biomass extraction and saponification of the lipid crude extract, 4-Me-6,8 – 16:2 was purified using urea inclusion and liquid chromatography on AgNO3-impregnated silica gel. The CFA structure was deduced from 1H-NMR and IR analyses confirming the trans configuration of double bonds. When tested against MCF-7 breast cancer cell line, 4-Me-6E,8E-16:2 reduced the viability in a dose dependent manner (up to 75% inhibition after 48h at 100µM). Numerous studies have demonstrated that cell proliferation is associated with hyperactivity of lipogenesis [3, 4]. We have then assessed the effect of 4-Me-6E,8E-16:2 on gene expression of two lipogenic enzymes, the ACC (Acetyl coA Carboxylase) and the FAS (Fatty Acid Synthase). At 50µM and after 24h, expression of ACC and FAS were reduced of 50% and 35%, respectively. These results are promising and need to be completed in order to determine the mechanisms by which 4-Me-6E,8E-16:2 reduce cancer cell proliferation and inhibits the expression of lipogenic enzymes.

The unique liquid chromatographic properties of Group 11 transition metals for the separation of unsaturated organic compounds

Silver(I) and copper(I) are known to form reversible complexes with π bonds, which have been exploited in LC for separating unsaturated organic compounds. Prominent examples include the use of AgNO3-impregnated silica gel in LC, and the use of copper(I) salts for selective extraction of alkenes from hydrocarbon mixtures. The Dewar-Chatt-Duncanson model is often invoked to explain the interaction between Ag(I) and Cu(I) and π bonds. However, it is unclear if such a reversible interaction is directly related to their d(10) outer electronic configurations. Particularly, Au(I) has not been reported to separate olefins with different numbers of double bonds in LC. Also, there has not been a systematic comparison of the liquid chromatographic properties of other d(10) transition metal salts (e.g., Zn(II), Cd(II)), making it difficult to fully understand the observed reversible interactions of Ag(I) and Cu(I) with π bonds. We demonstrate for the first time that silica gel impregnated with all three Group 11 transition metals with 1+ oxidation state strongly and similarly retain olefin compounds in LC, while transition metals from Groups 10 and 12 do not. We also tested a range of functionalized silica gels to improve the stability of Cu(I) and Au(I) ions on the surface of the silica.

Mating Sequence and Evidence for the Existence of a Female Contact Sex Pheromone in Brontispa longissima (Coleoptera: Chrysomelidae)

To clarify the presence of a pheromone in Brontispa longissima (Coleoptera: Chrysomelidae), we observed its mating behavior and conducted a series of bioassays. When male and female B. longissima came in contact, the male was observed to hold and mount the female, extend its penis toward her abdominal tip, and copulate. The male was observed to hold the female only after he had touched her with his antennae and/or forelegs. Males showed similar mating attempts toward females killed by freezing. On the other hand, no males showed mating attempts toward females washed with hexane, but they did toward females washed with hexane and re-treated with hexane extract of the female body or female elytra. Furthermore, males showed similar mating attempts toward a glass dummy treated with a solvent extract of female elytra. These results indicate the presence of a female sex pheromone that is perceptible by direct contact and plays an important role in mating in B. longissima. When the crude extract of female elytra was subjected to silica gel chromatography, only the fraction eluted with hexane elicited mating behavior in males. Furthermore, when the active fraction was chromatographed on AgNO3-impregnated silica gel, only the fraction eluted with hexane elicited mating behavior in males. The contact sex pheromone of B. longissima is therefore believed to consist of one or more less-polar compounds, probably saturated hydrocarbons.

A Photochemical Synthesis of Functionalized trans-Cyclooctenes Driven by Metal Complexation

Described is a scalable procedure for driving photochemical sytheses of trans-cyclooctene derivatives through metal complexation. During photoirradiation, reaction mixtures are continuously pumped through a column of a AgNO3-impregnated silica gel. The trans-cyclooctene derivative is selectively retained by the AgNO3-impregnated silica, but the cis-isomer elutes from the column back to the reaction flask, where it is photoisomerized and recirculated through the column. The method provides access to a range of usefully functionalized derivatives of trans-cyclooctene, including a derivative of 5-aza-trans-cyclooctene that underwent transannular cyclization upon treatment with bromine. The alkene stereochemistry is transferred with high fidelity to the hexahydropyrrolizine framework in the transannular cyclization.

COX-2 Inhibitory Activity of Cafestol and Analogs from Coffee Beans

Two kaurane diterpenes, namely cafestol (1) and kahweol (2), were isolated from hydrolyzed fraction of the fixed oil of Ethiopian Coffea arabica unroasted beans, using AgNO3-impregnated silica gel chromatography. In addition, cafestol palmitate (3) and kahweol palmitate (4), the two natural diterpene esters of C. arabica, were also synthesized. Compounds 1–4 were evaluated for anti-inflammatory activity using cyclooxygenase-2 (COX-2), cell aggregation, cell proliferation, cell adhesion, iNOS and antioxidant assays. The COX-2 inhibitory activity of cafestol (1) was found to be 20-fold more potent than kahweol (2) (IC50 value 0.25 μg/mL vs. 5.0 μg/mL), while compounds 3 and 4 were weakly active. The isolation and structure elucidation of the diterpenes 1 and 2, preparation of compounds 3 and 4, and their biological activities are presented in this paper.

SEPARATION OF STEROLS AND TRITERPENE ALCOHOLS FROM UNSAPONIFIABLE FRACTIONS OF THREE PLANT SEED OILS

Preparative HPLC was used to separate sterols and triterpene alcohols from the unsaponifiable matters in plant oils from Camellia weiningensis L., Brassica juncea L. and Microula sikkimensis. The isolated sterols and triterpene alcohols were acetylated and further purified by AgNO3 impregnated silica gel preparative thin layer chromatography (TLC). The isolated acetyl derivatives of sterols and triterpene alcohols were identified by melting point, specific rotation, infrared and mass spectrometry. The sterols were brassicasterol, campesterol, stigmasterol, β-sitosterol and Θ5-avenasterol, Θ7-avenasterol, Θ7-stigmastenol and α-spinasterol. The triterpene alcohols were cycloartanol, cycloartenol, 24-methylenecycloartanol, cyclobranol, dammaradienol, tirucalla-7,24-dienol, butyrospermol, β-amyrin, germanicol, α-4-taraxasterol and lupeol.

Host-location kairomone fromPeriplaneta americana (L.) for parasitoidAprostocetus hagenowii (Ratzeburg)

Chemically mediated host location in the eulophid parasitoidAprostocetus hagenowii (Ratzeburg) was investigated. In Y-tube bioassays 77.6% of female parasitoids responded to aPeriplaneta americana (L.) ootheca; parasitoids did not respond to air with no volatile stimuli. Frass from adult cockroaches was as attractive as an ootheca. Bioassay of one ootheca equivalent of five lipid fractions (eluted with hexane and 1, 5, 10, and 30% ether in hexane) from silica gel column chromatography indicated that the active component was a hydrocarbon. Further separation and bioassay of oothecal hydrocarbons by AgNO3-impregnated silica gel column chromatography indicated that the biological activity was in one fraction. Gas chromatographic (GC) analysis of this fraction revealed a single peak; this peak was identified by researchers in 1963, 1969, and 1972 as (Z,Z)-6,9-heptacosadiene. Qualitative and quantitative GC analyses of total hydrocarbons from oothecae, frass, and adult females were essentially identical; 6,9-heptacosadiene was the dominant hydrocarbon from each source. The alkadiene was 37 times more abundant in frass than on the ootheca. The volatilization of the alkadiene from oothecae was demonstrated by aeration and trapping on Super Q adsorbent. The current study is the first evidence for biological activity of (Z,Z)-6,9-heptacosadiene, a major hydrocarbon component on adult female American cockroaches, on their oothecae, and in their frass.

Sterol composition of Linneus torquatus (Nemertini) Anopla

The sterol fraction from the marine worm Linneus torquatus Coe (phylum Nemertini, class Anopla, family Lineidae) has been isolated, separated by HPLC and preparative TLC on AgNO 3-impregnated silica gel, and sterols identified using GC, GC-MS and NMR spectroscopy. It was shown that the fraction contains at least 12 sterols belonging mainly to Δ 5,22, Δ 5,24(28) and Δ 5 series. The major sterol components were 24-methylcholesta-5,24(28)-dien-3β-ol, cholesta-5,22E-dien-3β-ol, 24-nor-cholesta-5,22-dien-3β-ol and cholesterol.

疎水性銀添吸着剤によるヨウ素吸着

Hydrophobic adsorbents, which consist of porous styrene-divinylbenzene copolymer (SDB) impregnated with silver, were developed for the removal of iodine from the dissolver off-gas (DOG). The adsorption of iodine in a simulated off-gas including iodine, NOx and water vapor was examined by use of an adsorption column packed with the hydrophobic adsorbents.Silver impregnation methods using organic solutions were proposed. By use of dioxan and butylamine, which can swell the SDB easily, silver nitrate and metallic silver were uniformly distributed in the SDB particles.The breakthrough of iodine was not influenced by the presence of NOx and water vapor. For a macroporous SDB with the pore volume of 1.59ml/g-SDB and the average pore diameter of 500A, impregnated with metallic silver at silver content of 28wt%, a high adsorption capacity of 0.14g-I2/cm3-adsorbent was obtained, compared to that of a commercial adsorbent, AgNO3-impregnated silica gel. The impregnated silver was utilized about 91% for the iodine adsorption.

Separation of 20∶4n−6 and 20∶4n−7 by capillary gas‐liquid chromatography

The use of a capillary column coated with 100% cyanopropyl polysiloxane (CP(TM)Sil 88) allows the separation of several fatty acids associated with fat deficiency. Starting with liver mitochondrial phospholipids of weanling rats fed a fat-free diet, an unusual fatty acid was isolated, along with 20:4n-6, by thin-layer chromatography on AgNO3-impregnated silica gel plates. After partial hydrazine reduction of these acids, the resulting monoenes were isolated and subjected to ozonolysis in BF3/methanol. The resulting monomethyl and dimethyl esters were identified by gas chromatography/mass spectrometry. Our data indicate that the unusual component corresponds to 20:4n-7. Based on published biochemical and analytical studies and on our own chromatographic retention data, some of the other unusual fatty acids were tentatively identified as 18:2n-7, 20:2n-7 and 20:3n-7. The CP(TM)Sil 88 column appears to be a simple and useful tool for the separation of fatty acids of the palmitoleate series.

Preparative isolation of (+)-beta-eudesmol fromAmyris balsamifera

Optically pure (+)-beta-eudesmol is a possible starting material for the synthesis of several termite defense compounds. A two step procedure for the isolation of gram quantities of (+)-beta-eudesmol from commercially availableAmyris balsamifera oil (syn. West Indian sandalwood oil), containing 8% beta-eudesmol, was developed. Step one consisted of an efficient vacuum distillation of the total oil. Step two was a medium pressure LC separation with an AgNO3 impregnated silica gel stationary phase. Several other separation procedures failed due to the presence of many closely related sesquiterpene alcohols (75% of the oil).

Charakterisierung der Triglyceridmuster von pflanzlichen und tierischen Fetten sowie Human- und Tierseren mittels HPLC nach Vortrennung an AgNO3-impr�gnierten Kieselgel-Minis�ulen

A method is described that enables separation of complex triglyceride mixtures like those occurring in blends of vegetable fats, animal depot fats and serum lipids. By combination of adsorption chromatography on AgNO3-impregnated silicagel minicolumns and subsequent HPLC four characteristic triglyceride patterns can be obtained for each sample. The separation achieved reflects the degree of saturation and the chain length of fatty acids occurring in the triglyceride mixture. The isocratic HPLC is carried out with Hypersil ODS (5 μm) using propionitrile as eluent. The substances are detected with a sensitive differential-refractometer. 1 ml serum or 500 μg fat or oil are sufficient for a full description of the triglyceride patterns. Using this procedure 56 triglycerides could be characterized or identified by determination of their ECN-,t rel-values and their fatty acid moieties. The method is applicable for the detection of adulteration, blending and hydrogenation of fats and oils and can be used especially in physiological research due to its low sample need.

Preparation and spectroscopic characterization of molecular species of brain phosphatidylserines

This study describes the first preparation and spectroscopic characterization of naturally occurring phospholipids separated according to degree of unsaturation. Phosphatidylserines (PS) have been prepared from bovine brain and shown to be pure by extensive thin layer chromatographic analysis as well as by infrared spectroscopy and fatty acid analysis. The PS has been separated according to degree of unsaturation and prepared using AgNO 3-impregnated silica gel H thin-layer chromatography. Fatty acid analysis of the two principal PS subfractions indicates that they are enriched in the molecular species 1-octadecanoyl-2-docosahexaenoyl- sn-glycero-3-phosphorylserine and 1-octadecanoyl-2-octadecenoyl- sn-glycero-3-phosphorylserine. The identity of the two PS subfractions was further verified by rechromatographing on several thin layer systems and by infrared spectroscopy. With the use of a 100 MHz Fourier transform nuclear magnetic resonance (NMR) spectrometer, the spectra of bovine whole brain, white matter, gray matter, monoenoic, and hexaenoic PS were obtained. Distinct proton resonances were assigned to double bond protons, protons adjacent to a double bond, and protons between two double bonds, using fatty acid methyl ester standards. The various PS preparations gave different intensities of the various proton resonances which correlated with differences in fatty acid composition. The method provides a convenient, non-destructive spectroscopic method for distinguishing monoenoic and polyunsaturated species of intact phospholipids. Electron spin resonance studies of nitroxide-labelled cholestane in sonicated PS vesicles showed greater probe motion as the unsaturation of the acyl chains was increased. The hexaenoic PS vesicles were more fluid than monoenoic PS vesicles at all temperatures in the range 10–55°C. These results suggest that neuronal membranes are more fluid than myelin membranes as neuronal membranes contain more hexaenoic phospholipids.

Different utilization of arachidonic and dihomogammalinolenic acids by human platelet prostaglandin synthetase

Arachidonic and d ihomogammal inolenic acids were purchased by Nu Check Prep and [1-14C]-acids by Amersham. Specific radioact ivi ty of [1-14C]-20:40)6 and 20:3o)6 was 0.13 mCi/mmole . Pros taglandins were a generous gift of Dr. J. Pike (Upjohn and Co.). All organic solvents provided f rom Merck. Platelets were obtained f rom heal th donors 'who had not taken any drug ten days before vein punction. Platelets were isolated and 'washed according to a publ ished method [8]. After incubat ion of 'washed h u m a n platelets (4 ml at 109 pla te le ts /ml) 'with substrate (0.5 mM of 20:40)6 or 20:30)6) at different t imes, incubates were extracted and lipids were purified wi th a described radiochemical method [9]. Rf of PGs (PGFu : 0.09, PGE : 0.18, PGD : 0.46) vcere always visualized by addit ion of these unlabel led s tandard moIecules. Rf of th romboxane B1 is identical to that of th romboxane B~. We verified tha t th romboxane B1 measured on our plates is not a th romboxane B2 provided f rom 20:30)6 t rans formed in 20:40)6 in platelets. To do this verification, methanol extract of plate at Rf of th romboxanes B, ~vas submit ted to AgNO3-impregnated silicagel prepared by dipping into a 10 per cent methanol ic solution of AgNO3 during one minute. Eluent ~vas an organic phase of ethylacetate-acetic ac id-methanolisooctane-'water 110:30:35:10:100 [10]. In these condit ions Rf of s tandard th romboxane B2 is : 0.37, tha t of biosynthet ized th romboxane B1 is : 0.66 (Rf of others PGs are : PGF~ : 0.78 ; PGF~2 : 0.44 ; PGFla : 0.54 ; PGF._,u : 0.21 ; PGD1 : 0.71 ; PGE2 : 0.41). Moreover, monohydroxy acids produced f rom 20:3o)6 (12 L-hydroxy 8, 10-heptadecadienoic and 12 L-hydroxy 8, 10, 14-eicosatrienoie acids) ~vere scrapped off silver ni t rate silicagel plates [9] at Rf : 0.82 and 0.69 respectively. Pros taglandins endoperoxides 'were t rapped for reduction into PGFa by s tannous chloride [ l l , 12]. [14C]-PGF~a f rom endoperoxides one and [14C]-PGF~c~ f rom endoperoxides t~vo were determined by the radiochemical assay described [9]. Malondialdehyde de terminat ions ~vere done by the method of Flower et al. [13]. All the data sho~vn on figures represent the mean of three exper iments wi th different samples.

The separation of steryl acetates by thin-layer chromatography

The improved separation of steryl acetates using a nonpolar solvent system (1% ether in petroleum) with a continuous development technique is described. Silica gel, aluminium oxide, AgNO 3-impregnated silica gel, and Anasil B were used as adsorbents. The latter material afforded a separation of (24 R)- and (24 S)-isomers of 24-methylsterols.

The distribution of molecular species of phosphatidylinositol in ox brain and its subcellular fractions

1. The phosphatidylinositol content of white and grey matter of ox cerebral hemispheres did not differ. The phosphatidylinositol from grey matter was slightly enriched in palmitic acid and arachidonic acid, and that from white matter was enriched in eicosatrienoic (C(20:3)) acid. These regional differences were apparently due to the greater content of myelin in the white matter, since the same tendencies were observed when combined myelinic and non-myelinic subcellular fractions prepared from the cerebral hemispheres were compared. 2. Purified phosphatidylinositol was converted into its triacetylated methylated derivative and resolved to its molecular species by t.l.c. on AgNO(3)-impregnated silica gel. The tetraenoic molecular species was predominant in phosphatidylinositol from ox cerebral hemispheres, and this feature characterized all the phosphatidylinositol samples extracted from its regions or subcellular fractions. The grey matter was more enriched in the tetraenoic species and the white matter in the trienoic species. 3. The molecular-species composition of phosphatidylinositol from the subcellular fractions of ox cerebral hemispheres was studied. The trienoic species constituted nearly one-fifth of the phosphatidylinositol from two myelinic fractions. ;Large myelin' was more enriched in this species than was ;small myelin'. Both fractions also contained greater concentrations of the dienoic species than the non-myelinic subcellular fractions. The latter fractions, one containing nuclei and the other nerve endings plus mitochondria, were enriched in the monoenoic and tetraenoic species of phosphatidylinositol. The post-mitochondrial supernatant exhibited a pattern of distribution of phosphatidylinositol species intermediate between the myelinic and non-myelinic fractions.

Extraction of prostaglandin from AgNO3-impregnated silica gel layers for biological assay.

Different methods are compared for extraction of prostaglandins as free acids from AgNO3-impregnated silica gel layers for biological assay. A simple and quick method is described which gives 80–100% recovery of prostaglandins E1, E2, F1α and F2α.