Silicic Acid Column Research Articles

미역 추출물로부터 충치 원인균, Streptococcus mutans에 대한 항균물질의 분리 및 동정

건조 미역 10.4 ㎏을 acetone으로 추출, 여과한 다음 액-액 분배, 각종 크로마토그라피를 통하여 충치원인균, Streptococcus mutans에 대한 항균물질의 한 성분을 분리, 정제하고(160 ㎎, 수율 1.5×10?³%) 물질을 동정한 결과는 다음과 같다. 건조 미역 30 g 상당량의 추출액을 기준으로 용매 획분별 항균활성을 조사하였을 때, CHCl₃층이 73.2%로 가장 강하였고, hexane층이 62.0%이었으며, BuOH층이나 물층은 거의 없었다. 알루미나 칼럼에서는 산성 획분인 1% NH₄OH:MeOH(1:1)용매 획분이 81.0%의 항균활성을 나타내었으며, 실리카 칼럼에서는 CHCl₃:MeOH(95:5)용매에서 가장 높은 95.5%의 항균성을 나타내었고, ODS칼럼에서는 85% MeOH에서 96.4%의 항균성을 나타내었다. 최종적으로 ODS칼럼에서 95% MeOH를 이동상으로 하여 3개의 물질 S1(10 ㎎), S2(90 ㎎), S3(60 ㎎)을 분리 정제하였다. TLC에서 각 성분은 동일한 Rf값 0.42를 나타내어 동일한 물질로 추정되었으며, 이들을 메칠 유도체화한 성분들은 Rf값이 0.95로 바뀌어 이들 물질이 carboxyl기를 가지는 지방산으로 추정되었다. GC분석에서 표품 지방산과 비교한 결과, 이들은 C<SUB>18:4,n-3</SUB> 지방산과 retention time이 일치하였다. 또한, 메칠 유도체의 mass spectrum 분석 결과, m/z 290에 분자 이온 peak가 관측되어 C18:4,n-3 지방산의 methyl 유도체의 분자량과 일치하여, 이 물질을 3,6,9,12-octadecatetraenoic acid(stearidonic acid, C<SUB>18:4,n-3</SUB>) 지방산으로 동정하였다.

Possible Presence of Pheromone in Mating Behavior of the Pine Sawyer Monochamus saltuarius Gebler (Coleoptera: Cerambycidae)

The mating behavior of the pine sawyer, Monochamus saltuarius, is composed of two phases. The first phase is the female approach to pheromone-releasing males. When bioassayed by T-tube olfactometer, the female was moved toward the male and hexane extract of the male. The second phase is the male mounted the female to copulate after perceiving the contact pheromone being on the female body surface. When the hexane extract of the female body surface was applied onto a glass rod (dummy), the male showed the mating behavior to the dummy. The component of female body surface extract fractionated using a silicic acid column elicited mating behaviors of the males, and the other component by ether fraction was showed a high copulatory attempt. This study was discussed two-step functions of the pheromones in the beetle mating behavior.

Lipids isolated from bone induce the migration of human breast cancer cells

Bone is the most common site to which breast cancer cells metastasize. We found that osteoblast-like MG63 cells and human bone tissue contain the bile acid salt sodium deoxycholate (DC). MG63 cells take up and accumulate DC from the medium, suggesting that the bone-derived DC originates from serum. DC released from MG63 cells or bone tissue promotes cell survival and induces the migration of metastatic human breast cancer MDA-MB-231 cells. The bile acid receptor farnesoid X receptor (FXR) antagonist Z-guggulsterone prevents the migration of these cells and induces apoptosis. DC increases the gene expression of FXR and induces its translocation to the nucleus of MDA-MB-231 cells. Nuclear translocation of FXR is concurrent with the increase of urokinase-type plasminogen activator (uPA) and the formation of F-actin, two factors critical for the migration of breast cancer cells. Our results suggest a novel mechanism by which DC-induced increase of uPA and binding to the uPA receptor of the same breast cancer cell self-propel its migration and metastasis to the bone.

Functional and Structural Characterization of Rhodopsin Oligomers

A major question in G protein-coupled receptor signaling concerns the quaternary structure required for signal transduction. Do these transmembrane receptors function as monomers, dimers, or larger oligomers? We have investigated the oligomeric state of the model G protein-coupled receptor rhodopsin (Rho), which absorbs light and initiates a phototransduction-signaling cascade that forms the basis of vision. In this study, different forms of Rho were isolated using gel filtration techniques in mild detergents, including n-dodecyl-beta-D-maltoside, n-tetradecyl-beta-D-maltoside, and n-hexadecyl-beta-D-maltoside. The quaternary structure of isolated Rho was determined by transmission electron microscopy, demonstrating that in micelles containing n-dodecyl-beta-D-maltoside, Rho exists as a mixture of monomers and dimers whereas in n-tetradecyl-beta-D-maltoside and n-hexadecyl-beta-D-maltoside Rho forms higher ordered structures. Especially in n-hexadecyl-beta-D-maltoside, most of the particles are present in tightly packed rows of dimers. The oligomerization of Rho seems to be important for interaction with its cognate G protein, transducin. Although the activated Rho (Meta II) monomer or dimers are capable of activating the G protein, transducin, the activation process is much faster when Rho exists as organized dimers. Our studies provide direct comparisons between signaling properties of Meta II in different quaternary complexes.

Effect of starvation on lipid metabolism and stability of DHA content of lipids in horse mackerel (Trachurus japonicus) tissues.

For the purpose of characterizing the effect of starvation on 22:6n-3 (DHA) content in marine fish tissues, horse mackerel (Trachurus japonicus) were reared in a tank containing filtered, sterilized seawater under nonfeeding conditions for 107 d (survival rate of the fish was 96.51%). The crude total lipids (TL) of ordinary dorsal muscle, dorsal skin, and viscera of the starved individuals were separated into classes on silicic acid columns, and the constituents of the TL were quantified by gravimetric recovery from column chromatography. The TL, initially > 85% TAG in dorsal muscle, and even more in skin lipids, decreased dramatically within the first 44 d of starvation, and then decreased more gradually during the remainder of the test period, whereas the visceral TL decreased more slowly. The percentages of both saturated and monoenoic FA in the muscle TL also decreased somewhat, but those of DHA increased significantly in muscle during the test periods. Decreases in PE and PC initially were much smaller than TAG, but DHA levels remained high in both PE and PC. These findings indicate that all of the FA in the depot lipids of horse mackerel tissues are easily metabolized for energy production during starvation, but DHA in muscle lipids of the starved fish was maintained at a consistently high level, indicating that starvation did not affect DHA stability in phospholipids. The findings suggest that preservation of DHA in cell membrane lipid PE and PC is necessary for self-protection functions in starving fish.

Positional distribution of FA in TAG of enzymatically modified borage and evening primrose oils.

Stereospecific analysis was carried out to establish positional distribution of FA in the TAG of DHA, EPA, and (EPA + DHA)-enriched oils. In this study, TAG of enzymatically modified oils were purified using a silicic acid column. The TAG were then subjected to positional distribution analysis using a modified procedure involving reductive cleavage with Grignard reagent. The results showed that in DHA-enriched borage oil (BO), DHA was randomly distributed over the three positions of TAG, whereas gamma-linolenic acid (GLA) was mainly esterified at the sn-2 and -3 positions. In DHA-enriched evening primrose oil (EPO), however, DHA and GLA were concentrated in the sn-2 position. In EPA-enriched BO, EPA was randomly distributed over the three positions of TAG, similar to that observed for DHA. In EPA-enriched EPO, however, this FA was mainly located at the primary positions (sn-1 and sn-3) of TAG. In both oils, GLA was preferentially esterified at the sn-2 position. In (EPA + DHA)-enriched BO, EPA and DHA were mainly esterified at the sn-1 and -3 positions of TAG, whereas GLA was mainly located at the sn-2 position. In (EPA + DHA)-enriched EPO, GLA was mainly located at the sn-2 and -3 positions; EPA was preferentially esterified at the sn-1 and -3 positions, and DHA was found mainly at the sn-3 position.

Strategy and principles of the simultaneous analysis of prostanoids by gas chromatography/mass spectrometry/selected-ion monitoring

The simultaneous analysis of prostanoids (prostaglandins (PGs) and thromboxane (TX)) by gas chromatography/mass spectrometry/selected-ion monitoring (GC/MS/SIM) has been established for clinical specimens (plasma, urine, cerebrospinal fluid (CSF), and culture medium). Deuterated PGs and TX were used as internal standards for the isotope dilution assay method. They were derivatized to the methyl (Me) ester of the methoxime (MOX) dimethylisopropylsilyl (DMiPSi) ether form or the Me ester of the DMiPSi ether form by the same procedure. Samples were purified in three steps on an octadecyl silica column and a silicic acid column during the derivatization. The positive [ M−43] fragments of the PGs, TX, and their deuterated internal standards were monitored simultaneously. The calibration curves of each PG and TX were expressed linearly from 10 to 100 ng by the isotope dilution method. With this method, the PG and TX content of plasma, urine, CSF, and cell culture media were assayed in a single experiment.

Sulfonoglycolipids from the lichenized basidiomycete Dictyonema glabratum: isolation, NMR, and ESI-MS approaches.

Two distinct sulfonoglycolipid fractions were isolated from the basidiolichen Dictyonema glabratum by chromatography on Diethylaminoethyl (DEAE)-Sepharose, which resulted in rapid elution, followed by partition between aqueous sulfuric acid and an ethyl acetate-methanol-chloroform mixture and the content of the organic layer chromatographed of a column of silicic acid. The products were examined by nuclear magnetic resonance spectroscopy in their native rather than their acetylated forms, as in previous investigations. Each was methanolyzed to give the same methyl glycoside, Me-G. On electrospray ionization mass spectrometry (ESI-MS), it provided a pseudomolecular ion at m/z 303 in the positive-ion mode and a molecular ion at m/z 257 with a daughter ion at m/z 146 in the negative-ion mode, showing the presence of a sulfonate group S-linked to a hexosyl ring. An exclusively alpha-glucopyranosyl configuration was indicated by (1)H, (1)H correlation spectroscopy (COSY) and (1)H, (1)H total correlation spectroscopy (TOCSY). S-substitution occurred at CH(2)-6, because a high-field (13)C signal at delta 52.6 gave an inverted distortionless enhancement by polarization transfer (DEPT) signal and (1)H, (3)C heteronuclear multiple quantum coherence (HMQC) showed correlation with two H-6 signals. This 6-sulfono-alpha-quinovopyranoside group was present in the glycolipid fractions, O-alpha-D-Quip-6-sulfono-(1'<-->1)-2,3-diacyl-D-glycerol (polar fraction 1a; PF1a) and O-alpha-D-Quip-6-sulfono-(1'<-->1)-2- or -3-monoacyl-D-glycerol (polar fraction 1b; PF1b), the monoacyl derivatives not having been previously completely characterized in other systems. All components are typical of plant glycolipids. The most abundant fatty acid ester in PF1a and PF1b was C(16:0). Other esters present in PF1a were C(14:0), C(17:0), C(18:0), C(18:1) (oleic), C(20:0), C(21:0), and C(24:0), in contrast with C(14:0), C(17:0), C(18:0), and C(20:0) in PF1b. HMQC and TOCSY data can be used as fingerprints for detection of glycosylacylglycerides and sulfonoglycolipids and the positive ESI-MS ions at m/z 329 and 271 for identification of the latter.

Preparation of sn-2 long-chain polyunsaturated monoacylglycerols from fish oil by hydrolysis with a stereospecific lipase from &lt;i&gt;mucor miehei&lt;/i&gt;

The preparation of sn-2 eicosapentaenoyi glycerol and sn-2 docosahexaenoyi glycerol by the hydrolysis of fish oil by the sn-1, sn-3 stereo-specific immobilised lipase (Lipozyme IM-20) from mucor miehei is described. Monoacylglycerols obtained after the enzymatic hydrolysis were separated by silver nitrate-coated silicic acid column chromatography Both monoacylglycerols can be individually separated in almost pure form by elution from the column with a solvent mixture. The preparation of sn-2 substituted monoacylglycerols from marine origin allows their utilization as substrates for the synthesis of structured long-chain polyunsaturated fatty acid-containing triacylglycerols at specific positions.

Production of a hepatoprotective cerebroside from suspension cultures of Lycium chinense

Suspension cultures derived from Lycium chinense Miller seedlings produced significant amounts of a hepatoprotective cerebroside. Callus was induced from the stem of aseptic seedlings of L. chinense and maintained on MS solid media supplemented with 1.0 ppm 2,4-D and 0.1 ppm kinetin. Suspension cultures were established, and the cells were grown in the same liquid media in the dark. Lyophilized cells were extracted with a combined reagent of chloroform and methanol (2:1, v/v). An aqueous suspension of the evaporated cell extract was partitioned with chloroform, and the chloroform layer was subjected to silicic acid column chromatography followed by semi-preparative reverse phase C8 high pressure liquid chromatography. The purified compound showed hepatoprotective activity comparable to that shown by silymarin, and the structure was identified as 1-O-(β-D-glucopyranosyl)-(2S,3R,4E,8Z)-2-N-2'-hydroxy-(palmitoyl)-4,8-sphingadiene on the basis of spectral data. The content of the compound in cultured cell was tenfold higher than that of the fruit of L. chinense. The biosynthesis of the compound in cultured cell systems appears to parallel cell growth.

Determination of phospholipids in vegetable oil by fourier transform infrared spectroscopy

AbstractA method was developed to determine the total phospholipid content in vegetable oil by Fourier transform infrared spectroscopy (FTIR). Calibration curves of I‐α‐phosphatidylcholine (PC), I‐α‐phosphatidylethanolamine (PE), and I‐α‐phosphatidylinositol (PI) in hexane were generated at different concentrations. The optimal phospholipid absorption bands between 1200–970 cm−1 were identified and used for quantitative determination. High R2≥0.968 were observed between band areas and phospholipid standard concentrations. Phospholipids from crude soybean oil were obtained by water degumming, and purification was performed on a silicic acid column. The phospholipid contents of purified phospholipid extract, degummed and crude soybean oil determined from calibration equations were &gt;90, 0.0113, and 1.77%, respectively. High correlations of determination (R2≥0.933) were observed between the FTIR method and thin‐layer chromatography‐imaging densitometry method for the determination of phospholipid content. FTIR was found to be a useful analytical tool for simple and rapid quantitative determination of phospholipids in vegetable oil.

Simultaneous quantitation of ceramides and 1,2-diacylglycerol in tissues by Iatroscan thin-layer chromatography-flame-ionization detection.

Ceramides and 1,2-diacylglycerol have been demonstrated in intracellular signaling pathways. A method of simultaneous mass determination of ceramides and 1,2-diacylglycerol in tissues was developed using the latroscan which combines thin layer chromatography and flame ionization detection (TLC/FID) techniques. Because of relatively low amounts of these components in tissues, the fraction of nonpolar lipids, which included ceramides and glycerides, was eluted with chloroform/acetone mixture (3:1, vol/vol) through a silicic acid column to eliminate the polar phospholipids. Development of Chromarods was carried out using three solvent systems in a four-step development technique. The relationship of the peak area ratio to weight ratio compared with cholesteryl acetate added as an internal standard was linear. The amount of ceramides increased with incubation of rat heart homogenate and human erythrocyte membranes in the presence of sphingomyelinase (E.C. 3.1.4.12). The latroscan TLC/FID system provided a quick and reliable assessment of ceramides and 1,2-diacylglycerol.

Prostanoid synthesis in whole blood cells from fish of the Arabian Gulf

The ability to synthesise prostaglandins and thromboxane from 14C-labelled arachidonic acid was investigated in 11 species of fish from the Arabian Gulf. Cyclooxygenase activity was assessed in washed whole blood cells. Arachidonic acid and its metabolites were extracted and separated on silicic acid columns and thin layer chromatography (silica gel G). Total capacity to convert [ 14C]arachidonic acid to prostanoids varied from 1 to 35% among the 11 fish species studied. Gray shark ( Chiloscyllium griseum) blood cells had the highest capacity (37±0.4%) to convert arachidonate into prostanoids and two species of catfish ( Arius bilineatus and A. thalassinus) exhibited greater than 10% capacity to convert [ 14C]arachidonate into prostanoids. The major prostanoid synthesised by the two catfish ( A. bilineatus and A thalassinus) was 6-keto PGF 1α, a stable metabolite of prostacyclin, PGI 2. In contrast, A. teunispinis synthesised thromboxane B 2, a stable metabolite of thromboxane A 2. Thromboxane B 2 (TXB 2) was the major product synthesised by all three species of shark studied ( Chil. griseum, Carcharhinus plumbeus, Carch. melanopterus), with 6-keto PGF 1 α a minor product. Other fish studied showed a varied pattern of prostanoid synthesis. The synthesis of these prostanoids was almost completely blocked by preincubation of the whole blood cells from catfish and shark with indomethacin (0.5 μM) suggesting the involvement of cyclooxygenase-mediated prostanoid synthesis.

Isolation and Structural Identification of Sucrose Esters from Corn Spurrey ( Spergula arvensis ); Inhibition of Seed Germination

Polar as well as nonpolar extracts of Spergula arvensis (corn spurrey) were shown to cause inhibition of seed germination. The polar methylene chloride extracts were subjected to solvent partitioning and separations by a combination of silicic acid and Sephadex LH-20 column chromatography. All chromatographic fractions were monitored by a proso-millet seed germination assay, which resulted in the isolation of a group of sucrose esters (SE). The identity of the ester acids was determined by gas chromatography (GC) of their butyl esters. Further characterization of the SE by GC-mass spectrometry of their trimethylsilyl derivatives showed the presence of four different SE groups with two to seven acyl groups attached to sucrose. The SE isomers in greatest abundance contained either octanoic or dodecanoic acid along with two smaller branched-chain acids (butanoic and pentanoic acids). All four SE groups were inhibitory to seed germination. This is the first report of SE outside the family Solanaceae.

Successive Isolation and Separation of the Major Lipid Fractions Including Gangliosides from Single Biological Samples

Currently available techniques concerning extraction and characterization of the different lipids from biological specimens are designed for particular families and do not address consecutive isolation of lipid constituents in their globality. We describe here a simple, nondestructive chromatographic procedure that allows efficient elution and further analysis of the major lipid classes (neutral lipids, phospholipids, nonsialylated sphingolipids, and gangliosides) in their natural states from the same starting material. The procedure describes the use of solvent mixtures adapted to silicic acid column chromatography and permits 90–97% recovery of each of the above lipid groups. We have particularly concentrated on optimizing the efficient recovery of the diverse minor forms of gangliosides, free of other contaminants, from relatively small amounts of neural tissue. As model systems we have usedin vivoandin vitropreparations of mammalian retina for which only fragmentary data are available on lipid composition. We show that relative to brain, retina contains, for example, twofold more sphingomyelin and sixfold more GD3 ganglioside. In turn, cultured retinal glial cells contain twofold higher levels of globoside and eightfold higher amounts of GM3 ganglioside with respect to intact retina. Compared to previously published techniques, we obtain improved total ganglioside recovery, with enrichment of poly-sialogangliosides. The technique presented here should be widely applicable to analyze global lipid composition of diverse biological samples.

Studies on the Allelopathic Effect of Corn Spurry (Spergula arvensis L.) on Cole Crops and English Pea

These studies were initiated to investigate severe growth inhibition observed when some vegetable crops were infested with corn spurry (Spergula arvensis L.). Interference by a natural population of the weed reduced the shoot weights of English pea (Pisum sativum L.) and collard (Brassica oleracea L.) by 93% and 72%, respectively. In a greenhouse experiment where light competition by corn spurry was prevented, broccoli (Brassica oleracea L.) shoot weights were reduced by corn spurry, but pea weights were not different from the controls. Homogenized corn spurry shoot tissue incorporated into a greenhouse potting medium inhibited the growth of both species, and a concentration effect was observed. Sequential hexane, dichloromethane, methanol, and 50% aqueous methanol extracts of corn spurry root and shoot tissue were tested for inhibitory activity using millet seed germination and broccoli seedling growth bioassays. Dichloromethane, methanol, and aqueous methanol shoot extracts were inhibitory to broccoli; whereas all shoot extracts inhibited millet germination. Shoot extracts were more inhibitory than root extracts. Further fractionation of the inhibitors using a combination of reversed-phase sephadex LH-20 and silicic acid column chromatographic procedures showed that a major portion of the millet germination inhibition was due to sucrose esters (SE). Preliminary characterization of the esters showed that there were four different SE groups. The major groups contained either octanoic or dodecanoic acid along with butanoic and petanoic acids. All groups inhibited seed germination at concentrations as low as 20 ppm. This is the first report of the SE class of defense chemicals in plant species outside of the solanaceae family.

Low molecular weight storage material in infantile ceroid lipofuscinosis (CLN1).

The identification of the genetic defect in CLN1 as a palmitoyl-protein thioesterase deficiency initiated a search for the lysosomal storage material. Pulse-chase labelling of fibroblasts and lymphoblastoid cell lines with [35S]cysteine revealed the presence of lipid [35S]cysteine material in CLN1 fibroblasts and not in controls, CLN2 or CLN3 patients or other patients with lipidosis. A single band comigrated with the acylcysteine standard and labelling with [3H]palmitate showed a band of material which eluted from the silicic acid column with the phospholipid fraction and which co-migrated with the lipid-[35S]cysteine band. The storage material is tentatively identified as palmitoylcysteine.

The use of a classic lipid extraction method for simultaneous recovery of organic pollutants and microbial lipids from sediments

An analytical protocol is described which allows for the determination of polycyclic aromatic hydrocarbons (PAH) and phospholipid fatty acids on the same sample. This procedure allowed for the simultaneous evaluation of PAH concentration and its biological effects on the microbial community. The total extract was separated into PAH, neutral lipid, glycolipid and phospholipid fractions on a silicic acid column by sequential elution using hexane, chloroform, acetone and methanol. This method was shown to be simple, effective and reproducible, and can be applied to very small samples (20–100 mg of sediment). PAH concentration was determined directly from the hexane fraction by GC/MS analysis without further preparative steps. Microbial community structure was simultaneously determined using phospholipid ester-linked fatty acids (PLFA). This method was evaluated based on analysis of Standard Reference Material (NIST SRM 1941a), and applied to environmental samples from Eagle Harbor, Puget Sound, Washington.

Lipid peroxidation of mitochondrial membrane induced by D 1: an organic solvent extractable component isolated from a crude extract of burn eschar

D 1, an organic solvent extractable component of an extract from burn eschar was characterized and the mechanism of its inhibitory effect on mitochondrial respiratory functions was investigated. Using silicic acid column chromatography, it was found that D 1 consisted of two parts: a toxic simple lipid and a non-toxic complex lipid. Six subunits were obtained by further silicic acid column chromatography, among them, the no. 4 and no. 6 were toxic. Infrared spectrometric studies showed that no. 4 consisted of esters, while no. 6 were peroxides. D 1 also contained large amounts of malonaldehyde (MDA) and lipid hydroperoxides (ROOH). Vitamin E was found to prevent the inhibitory action of D 1 on mitochondrial function and to reduce the amount of MDA produced. However, if vitamin E was introduced after the addition of D 1, the inhibition could not be prevented, although there was still a reduction in MDA production; therefore, MDA per se was probably not the cuuse of the inhibition. D 1 induced ROOH formation in mitochondrial membranes. Cumene hydroperoxide, an organic hydroperoxide, was capable of inhibiting the mitochondrial function. This inhibitor action was blocked by vitamin E. It is speculated that ROOH in D 1 is probably the element that inhibits mitochondrial function. That D 1 caused lipid peroxidation of membrane lipid was also proved by analysing the fatty acid composition of mitochondrial membranes before and after treatment with D 1. It was found that the amount of polyunsaturated fatty acids decreased and that of saturated fatty acids increased after incubation with D 1, a direct proof that lipid peroxidation has occurred. Lipid peroxidation of the membrane lipid was the result of the action of oxygen free radicals. This process was best shown by a chemilumirtescence study. In this experiment, D 1 induced chemiluminescence which was dose-dependently related to the amount of D 1 used. This was probably the most direct proof that D 1 caused lipid peroxidation of membrane lipid resulting in damage of the mitochondria. Copyright

The analysis of PAHs nitrogen derivatives in the sewage sludges of upper Silesia, Poland

The dimethylformamide extracts from sewage sludges from a chosen sewage — treatment plant in highly industrialized region of Poland were investigated. Using the column chromatography with Al2O3 and silicic acid as the stationary phases, the fraction of azaarenes has been separated. GC-MS analysis of azaarenes fraction made it possible to identify 10 azaarenes and several of their alkyl derivatives, which dominate in the organic matter extracted from sewage sludges. The silicic acid column chromatography in connection with the reduction of nitroarenes and derivatization with pentafluoropropionic acid anhydride yielded an enriched nitrofraction. Nitroarenes present in this fraction as amide derivatives were also analysed by GGMS method. There was found presence of 3 nitroarenes and their methyl derivatives.