Silica Gel TLC Research Articles

Simultaneous Densitometric Determination of First Line Anti-TB Drugs in Binary, Ternary, and Quaternary Mixtures

This article presents the development of a new densitometric method for the simultaneous separation and determination of isoniazid (INH), ethambutol (EMB), rifampicin (RIF), and pyrazinamide (PZA). INH, RIF, and PZA were determined by UV detection at 270 nm, and then EMB was derivatized and detected at 250 nm. The studied drugs were determined in their pure forms, available pharmaceutical preparations, binary, ternary, and quaternary synthetic mixtures. The study was performed on precoated silica gel TLC plates using acetonitrile:chloroform:2-propanol:4 N ammonia as a mobile phase (34:31:34:1 v/v/v/v). The studied drugs have been satisfactorily resolved with Rf values 0.31 ± 0.01, 0.48 ± 0.03, 0.73 ± 0.02, and 0.09 ± 0.01 for INH, RIF, PZA, and EMB, respectively. The developed method has been validated according to ICH guidelines providing good correlation coefficients (0.9991–0.9998) with a detection limit of 18.14 to 94.34 ng/band for all studied drugs. The proposed method has the potential to determine INH, RIF, and PZA simultaneously (before spraying) and EMB after spraying of the plate with cobalt sulfate without any interference. The method is new, rapid, and inexpensive compared to the official and reported methods for the assay of the quaternary mixture of the studied drugs.

Di-rhamnolipid is a mosquito pupicidal metabolite from Pseudomonas fluorescens (VCRC B426)

Pseudomonas fluorescens Migula (VCRC B426) produces a secondary metabolite, which was found to be active against pupae of vector mosquitoes namely Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti. The mosquito pupicidal metabolite from P. fluoescens was mass produced and separated by ethyl acetate extraction and purified further by silica gel column chromatography, FPLC, HPLC and TLC. The purified metabolite was characterized by NMR, FT-IR, LC–MS and MALDI-TOF. The FT-IR, 1H and 13C NMR results showed that it is a rhamnolipid (di-rhamnolipid). The matrix assisted laser desorption and ionization-time-of-flight spectrum of the sample showed predominant pupicidal component produced by P. fluorescens was the molecule mass of 673.40Da. Owing to its high toxicity to mosquito pupae, especially Anopheles sp., and Aedes sp., the di-rhamnolipd has potential in the control of the vectors of dengue, chikungunya, yellow fever and malaria. This is the first report of mosquito pupicidal di-rhamnolipid from P. fluorescens.

Discovery of a new caerulescent Psilocybe mushroom in Germany: Psilocybe germanica sp.nov.

Sixty years ago, the ethnomycologist R. G. Wasson discovered an ancient mushroom cult in Oaxaca, Mexico. The famousmycologist R. Heim classified the psychoactive mushroom species in the genera Psilocybe and the eminent natural product chemist Albert Hofmann published the isolation, structural elucidation and synthesis of the new alkaloids psilocybin and psilocin in 1958. These indole derivatives were also found in a number of other Psilocybe species encountered in Asia and Europe and it is now known that other psychoactive species exist in the genera Pluteus, Panaeolus, Inocybe andGymnopilus. Hallucinogenic mushrooms of the main genus Psilocybe are known from countries with a wet climate. Besides the cultivation of subtropical species such as Psilocybe cubensis (Earle) Singer, a range of other naturally growing species are commonly found in Europe. They deserve increasing attention because some species can yield up to thousands of fruiting bodies in relatively small spaces. The species Psilocybe semilanceata (Fr.) Kummer contains comparatively high amounts of psilocybin (around 1% dry weight) and some baeocystin, which is the monomethyl analogue of psilocybin, whereas the phenolic compound psilocin is only found in traces in these mushrooms (Figure 1). This small species usually grows in only scattered patterns on pasture. In recent decades, however, the psychoactive mycoflora of Europe has been transformed deeply. In 1946, E. Wakelfield described a new species from Kew Gardens (London, UK) that became known as Psilocybe cyanescens Wakefield. This mushroom typically exhibits strong blueing behaviour after bruising and also spontaneously during ageing. This species grows on wood chips and rich compost derived from plant residues. It has been suggested that these mushrooms grew adventitiously in botanical gardens following importation of plant material from foreign countries. In 1962, this species was also found in the Pacific Northwest, USA and Kubicka first discovered other blueing mushrooms in woods near Sazava (Czech Republic) in 1942. The species was named Psilocybe bohemica Sebek and further descriptions are available in the Czechmycological literature. Krieglsteiner shed new light a variety of mycological collections obtained from wood debris about 30 years ago It is now clear, however, that Psilocybe cyanescens was not in this collection. One of the present authors (J. G.) has studied these mushrooms since 1986 and found genuine Psilocybe bohemica in the Czechoslovakia in 1986 and later in 1989. It appears that the distribution of Psilocybe cyanescens has been expanding in Germany, and possibly other European countries, during the last 30 years. The reason for

New Cembranoid Diterpenes from Sarcophyton trocheliophorum

Aims: To isolate the unpolar bioactive diterpenes in pure forms from the soft coral Sarcophyton trocheliophorum, collected from Red Sea at the Egyptian coasts, and to identify their structures using diverse spectroscopic means (NMR [1D&2D] and MS). Additionally, to study the antimicrobial and cytotoxicity of the isolated compounds compared with the main crude extract of the soft coral. Study Design: Collection of the soft coral, Sarcophyton trocheliophorum from Red Sea at coast of Hurgada, east Egypt, its homogenization, maceration in suitable organic solvent for extraction of the desired bioactive compounds and their purification by a series of different chromatographic techniques, identification of the compounds by mass spectrometry and NMR (1D&2D) spectroscopy, and then determination of the antimicrobial and cytotoxicity for them compared with their original crude extract. Place and Duration of Study: Institute of Organic and Biomolecular Chemistry, University of Gottingen, Germany; and Chemistry of Natural Compounds Department, National Research Centre, Egypt, between August, 2008 and December, 2010. Methodology: The soft coral Sarcophyton trocheliophorum was homogenized in a blender, macerated with chloroform-methanol, and the chloroform layer was concentrated in vacuo; while aqueous methanol solution was re-extracted with n-butanol, and the latter was concentrated to dryness. The resultant whole crude extract was fractionated using several subsequent column chromatographies on silica gel, and the fast fractions containing the unpolar components were purified by different column chromatographies (silica gel, Sephadex column and preparative TLC) to deliver the desired diterpenes in pure form. Structures of the afforded diterpenes were identified by mass spectrometry (ESI and HRESI-MS), NMR analysis (1H,13C NMR, and 2D NMR) and by comparison with reference data. The antimicrobial activity was determined by disc diffusion assay, while the cytotoxicity was determined using brine shrimp assay. Results: Two new unpolar diterpenes, cis-cembrene C (1) and cis-cembrenene C (2) were isolated along with (+)-sarcophine (3), (+)-sarcophytoxide (4) and (-)-sarcophytoxide (5), from the chloroform-soluble extract of soft coral Sarcophyton trocheliophorum, collected from Red Sea. Four additional unpolar sesquiterpenes; s-elemene (6), caryophyllene (7), alloaromadendrene (8) and 1-methyl-4-(5-methyl-1-methylene-hex-4-enyl)-cyclohexene; bisabolene (9) were identifed by GC-MS analysis. Structures of the new diterpenes 1-2 were identified by spectroscopic data (1H, 13C, 1H-1H COSY, HMQC, HMBC, HREI-MS and HRESI-MS) interpretation and comparison with related structures. The antimicrobial and cytotoxic activities of compounds 1-5 were evaluated in comparison with the original soft coral crude extract. Conclusion: The soft coral Sarcophyton trocheliophorum is a prolific source for production of numerous bioactive compounds with diverse structures and biological activities which can be exploited for their commercial production.

Bioactive Secondary Metabolites from Terrestrial Streptomyces baarnensis MH4

Aims: Isolation, screening and identification of potent bioactive compounds producing Streptomyces sp. from the terrestrial soil of Egypt. Fermentation study, product recovery, purification and its characterization was also taken into consideration. Study Design: Cultivation in lab fermenter, solvent extraction and purification of the compounds by column chromatography, identification of the compounds by mass spectrometry and NMR spectroscopy, determination of the antimicrobial activity. Place and Duration of Study: Institute of Organic and Biomolecular Chemistry, University of Gottingen, Germany; Department of Microbial Chemistry, National Research Centre, Egypt and Chemistry of Natural Compounds Department, National Research Centre, Egypt, between January, 2009 and December, 2010. Methodology: The strain was cultivated at 25-liter shaker culture and the fermented broth was filtered and the filtrate was extracted by XAD-16 resin and then by methanol-water, and the concentrated water residue was extracted by ethyl acetate followed by evaporation till dryness. The resultant crude extract was fractionated on silica gel and the components were purified by column chromatography (silica gel, Sephadex column and preparative TLC). The pure component was identified by mass spectrometry (ESI and HRESI-MS), NMR analysis (1H,13C NMR, and 2D NMR) and by comparison with reference data. The antimicrobial activity was determined by disc diffusion assay. Results: The morphological, biochemical and physiological characterization suggested that isolate MH4 belongs to the genus Streptomyces. Comparison the obtained data with literature data and showed 99% identity coverage towards Streptomyces baarnensis. The scale up fermentation of the isolate MH4 yielded eight known metabolites, nonactic acid (2), homononactic acid (3), 3-(3,3-Di-indolyl)propane-1,2-diol (4), turbomycin A (5), indolyl-3-acetic acid, indolyl-3-carbaldehyde, indolyl-3-carboxylic acid and 2'–deoxyadenosine. Structures of the isolated compounds were assigned by intensive studies of nuclear magnetic resonance (NMR) and mass spectrometry and comparison with corresponding literatures. The taxonomical characterization, fermentation, and biological activity of the Streptomyces baarnensis MH4 were investigated. Conclusion: The isolate Streptomyces baarnensis MH4 is a potent producer of several antibiotic compounds which can be exploited for their commercial production.

Eco-Friendly HPTLC Method for Simultaneous Analysis of Simvastatin and Ezetimibe in Pharmaceutical Preparations and Trying to Use Limonene as Eluent

In this study, a simple, rapid, sensitive and green High Performance Thin Layer chromatography (HPTLC) method was developed and validated for determination of Simvastatin and Ezetimibe in tablet dosage form. The method was carried out in TLC silica gel of nano particle size on glass plate 60 F 254, 10 cm ×10 cm. Two solvent systems were chosen according to the Green Analytical Chemistry (GAC) parameters. Acetone: Heptane: Isopropyl alcohol in the proportion of 10:10:5, (v/v/v) with Rf Value for Simvastatin and Ezetimibe was 0.513 and 0.312 respectively. The linear regression coefficients were 0.999 and 0.998 for Simvastatin and Ezetimibe with respect to peak area and height in the concentration range of 600 - 1500 ng/spot and 150 - 375 ng/spot respectively. To get greener solvent, heptane was replaced with limonene in the second elution system but this system could separate either Simvastatin or Ezetimibe solely in single preparations but could not separate both of them simultaneously because both of them has almost the same Rf. The linear regression coefficients were 0.998 and 0.995 for Simvastatin and Ezetimibe respectively with the same concentration range of the first system. TLC plates of nano sized particles offer sharper separations due to small particle size and narrow fractionation. Theoretical plate heights (h values) are considerably smaller than those of the standard TLC plate. In addition diffusion and – as a consequence -band broadening are much lower. Also shorter developing times and shorter migration distances: After only a few centimeters an optimal separation has been achieved. Smaller samples of 0.01-0.1 μl (10-100 nanoliters). The samples applied are considerably smaller than with standard plates, thus it is possible to apply a large number of samples to a very small surface area, without samples interfering with each other. Finally increased detection sensitivity (nanogram level, hence nano plate). With fluorescence evaluation pico-gram quantities can be detected

Blockage of the early step of lankacidin biosynthesis caused a large production of pentamycin, citreodiol and epi-citreodiol in Streptomyces rochei.

In our effort to find the key intermediates of lankacidin biosynthesis in Streptomyces rochei, three UV-active compounds were isolated from mutant FS18, a gene disruptant of lkcA encoding a non-ribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrid enzyme. Their structures were elucidated on the basis of spectroscopic data of NMR and MS. Two compounds of a higher mobile spot on silica gel TLC (Rf=0.45 in CHCl3-MeOH=20:1) were determined to be an epimeric mixture of citreodiol and epi-citreodiol at the C-6 position in the ratio of 2:1. In contrast, the compound of a lower mobile spot (Rf=~0 in CHCl3-MeOH=20:1) was identical to a 28-membered polyene macrolide pentamycin. The yields of citreodiols and pentamycin in FS18 were 5- and 250-fold higher compared with the parent strain. Introduction of a second mutation of srrX, coding a biosynthetic gene of the signaling molecules SRBs, into mutant FS18 did not affect the production of three metabolites. Thus, their production was not regulated by the SRB signaling molecules in contrast to lankacidin or lankamycin.

Chemical Constituents of the Roots of Trichosanthes kirilowii

The genus Trichosanthes Linn. (Cucurbitaceae) is composed of about 50 species distributed mainly in Southeast Asia. In traditional Chinese medicine the roots of Trichosanthes kirilowii Maxim., commonly known as “Tian Hua Fen,” are used for the treatment of cough, inflammation, polydipsia, and diabetes [1]. Most previous phytochemical studies of T. kirilowii have focused on the fruits and seeds, and triterpenoids, sterols, amino acid, lipids, and other compounds have been isolated from different parts of the plant [2–5]. In our research on chemical constituents of the roots of T. kirilowii, which has rarely been reported in recent years, 18 compounds were isolated and elucidated as 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid (1), allantoin (2), nicotinic acid (3), xanthine (4), adenine (5), uracil (6), thymine (7), guanosine (8), uridine (9), phenylalanine (10), tyrosine (11), 1-C-(p-hydroxyphenyl)-glycerol (12), furoic acid (13), p-hydroxybenzoic acid (14), p-hydroxybenzaldehyde (15), succinic acid (16), -spinasterol-3-O-D-glucopyranoside-6 -O-palmitate (17), and bryonolic acid (18). Plant Material. The roots of Trichosanthes kirilowii were collected from Hebei Province of China in July 2010 and authenticated by Prof. Tian-Xiang Li, Tianjin University of Traditional Chinese Medicine. A voucher specimen was deposited in the herbarium of pharmacognosy, Tianjin University of Traditional Chinese Medicine. Extraction and Isolation. The air-dried roots (14 kg) were cut into small pieces and refluxed with 95% EtOH and then with 50% EtOH twice. The extract was suspended in water and successively partitioned with petroleum ether (60–90 C), CH2Cl2, EtOAc, and n-BuOH in turn. The petroleum ether extract (45 g) was subjected to silica gel column chromatography eluting with petroleum ether–EtOAc in a gradient manner, and further purified by silica gel and Sephadex LH-20 column chromatography repeatedly to afford compounds 17 (80 mg) and 18 (25 mg). The CH2Cl2 extract (30 g) was subjected to silica gel column chromatography eluting with gradient CH2Cl2–EtOAc to get compounds 12 (8 mg) and 15 (6 mg). The EtOAc extract (18 g) was repeatedly separated by silica gel, Sephadex LH-20, and preparative TLC to yield compounds 2 (85 mg), 3 (5 mg), 4 (5 mg), 6 (72 mg), 9 (3 mg), 13 (7 mg), 14 (15 mg), and 16 (100 mg). The n-BuOH extract (170 g) was chromatographed over a D101 macroporous resin column eluting with H2O and 30%, 50%, 70%, and 95% EtOH. The 30% eluate (37 g) was subjected to silica gel column chromatography and further purified on Sephadex LH-20 and ODS columns to give compounds 1 (20 mg), 5 (20 mg), 7 (3 mg), 8 (150 mg), 10 (80 mg), and 11 (8 mg). The structures of the compounds were identified by a combination of spectroscopic methods (MS, 1H NMR, and 13C NMR) and physicochemical properties. The spectroscopic data of all the compounds were in good agreement with the literature data. 1H and 13C NMR spectra were recorded on 400 MHz and 600 MHz Bruker AV III spectrometers (Bruker, Switzerland). ESI-MS data were obtained on a Waters Quattro Premier XE mass spectrometer (Waters, USA). Silica gel (100–200 and 200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., China), Sephadex LH-20 gel (GE Healthcare Bio-Sciences AB, Sweden), ODS (YMC Co. Ltd, Japan), and D101 resin (Tianjin Haiguang Chemical Co. Ltd., China) were used for column chromatography. TLC was carried out using precoated plates with GF254 silica gel (Qingdao Haiyang Chemical Co. Ltd., China). Spots on TLC were visually observed under UV light and by spraying with 5% H2SO4 reagent followed by heating.

A unique dansyl-based chromogenic chemosensor for rapid and ultrasensitive hydrazine detection.

We developed a new dansyl phthalimide-based fluorescent chemosensor for hydrazine detection. Upon a Gabriel type-based hydrazinolysis of dansyl phthalimide (DPI) in the presence of hydrazine in a mixture of HEPES buffer (pH 7.0, 20 mM) and DMSO (1/9, v/v) at room temperature, the chemosensor produces fluorescent dansyl-NH2 with the maximum emission wavelength changed from 475 nm to 512 nm along with a color change from yellow to colorless, allowing colorimetric detection of hydrazine by the naked eye. DPI can selectively detect hydrazine over other environmentally abundant ions. Moreover, DPI coated with silica gel TLC plates could act as a visual and fluorimetric probe for hydrazine vapor at a partial pressure of 5.5 × 10-3 mm Hg over other potentially interfering volatile analytes, including hydrogen peroxide, ethylenediamine, urea, ammonium hydroxide and methylamine. DPI can also be used for the detection of hydrazine in water samples and HeLa cells without appreciable interference from other biologically abundant analytes. The limit of detection is 6.01 ppb (1.88 × 10-7 M), which is well below the accepted limit (10 ppb) for hydrazine set by the U.S. Environmental Protection Agency (EPA).

Amine Sensing with Distyrylbenzenes and Their Hexamethylene-Linked Polymers: Spraying Them On

We herein describe synthesis and property evaluation of two aldehyde-appended nonconjugated distyrylbenzene polymers as amine sensing platforms and compare their performance to that of their monomers. The monomers and polymers were dissolved in organic solvents and spray-deposited onto silica gel and neutral alox TLC plates so as to give small strip-shaped sensor arrays. With these, differential amine detection was achieved; we find that a neutral silica gel support is better able to discern the amines than alox TLC plates.

Chemical constituents from a Tibetan medicine Meconopsis horridula

A phytochemical investigation on the aerial parts of a Tibetan medicine Meconopsis horridula, by solvent extraction, repeated chromatographies on silica gel, Sephadex LH-20, and preparative TLC techniques, led to the isolation of 9 compounds. By spectroscopic analysis and comparison of its 1H and 13C-NMR data with those in literatures, their structures were identified as oleracein E(1), N-( trans-p-coumaroyl) tyramine (2), chrysoeriol (3), apigenin (4), hydnocarpin (5), p-coumaric acid glucosyl ester (6), stigmast-5-ene-3beta-ylformate (7), 3beta-hydroxy-7alpha-ethoxy-24beta-ethylcholest-5-ene (8), and beta-sitosterol (9), respectively, among which compounds 6-8 were isolated from the genus for the first time,and 1,3 were isolated from the species for the first time. A MTT method was applied to evaluate the cytotoxic activity of compounds 14 against the human hepatocellular liver carcinoma cell line (HepG2), and compound 1 showed significant cytotoxicity against HepG2,with its inhibitory rate of 52.2% at 10 micromol x L(-1).

Chemical investigation of the roots of Polygala sibirica L.

AimTo investigate the chemical constituents of the roots of Polygala sibirica L. (Polygalaceae) MethodThe isolation was performed by solvent extraction and various chromatographic techniques, including silica gel, Sephadex LH-20, ODS, semi-preparative HPLC, and preparative TLC. The chemical structures were elucidated based on extensive spectroscopic analysis, including HR-ESI-MS and 1D- and 2D-NMR spectroscopic data. ResultsA total of sixteen compounds, including five xanthones (5, 7–10), five saccharide esters (1, 3, 4, 12, 13), two flavonoids (14, 16), two triterpenoids (11, 15), one phenylpropanoid (6), and one benzophenone glycoside (2) were isolated. Their structures were determined as sibiricose A7 (1), sibiriphenone A (2), polygalatenoside A (3), polygalatenoside C (4), lancerin (5), 3, 4, 5-trimethoxycinnamic acid (6), 6-hydroxy-1, 2, 3, 7-tetramethoxyxanthone (7), 1, 3, 7-trihydroxy-2-methoxyxanthone (8), onjixanthone II (9), 1, 2, 3, 6, 7-pentamethoxyxanthone (10), presenegenin (11), 3′-O-3, 4, 5-trimethoxycinnamoyl-6-O-4-methoxy benzoyl sucrose (12), tenuifoliside C (13), 5, 3′-dihydroxy-7, 4′-dimethoxyflavonol-3-O-β-D-glucopyranoside (14), tenuifolin (15), and rhamnetin 3-O-β-D-glucopyranoside (16). ConclusionCompounds 1 and 2 are two new compounds from P. sibirica.

Flavonoids, Antioxidant and Antibacterial Activities of Eryngium triquetrum

The genus Eryngium belongs to the Apiaceae family (Umbelliferae), subfamily Saniculoideae, which consists of about 300 species [1]. Plants of this genus have shown various activities such as anti-inflammatory [2], antimalaria and antifungal [3], antioxidant and antimicrobial [4], antidiabetic [5], and analgesic properties [6]. Air-dried and powdered aerial parts (1.5 kg) of Eryngium triquetrum Vahl. were macerated at room temperature with MeOH–H2O (70:30, v/v) for 24 h three times. After filtration and concentration, the residue was dissolved in water (600 mL). The resulting solution was extracted successively with CHCl3, EtOAc, and n-butanol. Concentration in vacuo led to the following extracts: CHCl3 (0.8 g), EtOAc (1.3 g), and n-butanol (11 g). The butanolic extract of Eryngium triquetrum (11 g) was column chromatographed on polyamide SC6 with a gradient of toluene–MeOH with increasing polarity; Fr. 4 (70 mg), obtained from 85% toluene, was subjected to silica gel column chromatography eluted with EtOAc–MeOH–H2O (10:1:0.5), leading to two subfractions: Fr. 1 and Fr. 2. Subfraction 1 was separated by silica gel column chromatography eluted with CH2Cl2–MeOH (8:2) to afford compound 1 (10 mg). Subfraction 2 yielded compound 2 (60 mg), which was obtained as a yellow precipitate. Fraction 6 (70 mg), obtained from 80% toluene, was further subjected to silica gel column chromatography eluted with EtOAc–MeOH–H2O (10:1:0.5) to give compound 3 (40 mg). The ethyl acetate extract (1.3 g) was column chromatographed on silica gel with a gradient of CHCl3–MeOH with increasing polarity. Fraction 2 (30 mg) and Fr. 3 (35 mg), obtained from 95% and 90% CHCl3 successively, were separated by silica gel column chromatography eluted with EtOAc–MeOH–H2O (10:1:0.5) to afford compounds 4 (9 mg) and 5 (8.5 mg). The structures of compounds 1–5 were established by chemical and spectral analysis, UV, NMR, acid hydrolysis, and HPLC-UVDAD-MS, as well as by comparing their spectroscopic data with those reported in the literature. Acid Hydrolysis. The pure compounds were treated with 2M HCl at 100 C for 1 h. The hydrolysates were extracted with EtOAc, and the aglycons were identified by their UV spectra in methanol and by comparison of their Rf with authentic samples. Sugars were identified in the aqueous residue by comparison with authentic samples on silica gel TLC impregnated with 0.2 M NaH2PO4, solvent Me2CO–H2O (9:1), revealed with aniline malonate. Compound 1. C21H20O11. UV (MeOH, max, nm): 267, 347; +NaOH: 275, 325, 401; +AlCl3: 273, 398; +AlCl3/HCl: 273, 397; +NaOAc: 274, 377; +H3BO3: 268, 355. 1H NMR (250 MHz, CD3OD, , ppm, J/Hz): 8.09 (2H, d, J = 8.9, H-6 , 2 ), 6.91 (2H, d, J = 8.9, H-5 , 3 ), 6.42 (1H, d, J = 1.9, H-8), 6.22 (1H, d, J = 1.9, H-6), 5.28 (1H, d, J = 7.3, Glc H-1 ), 3.26–4.14 (sugar protons). This compound was characterized as kaempferol 3-O-D-glucoside [7]. Compound 2. C30H26O13. UV (MeOH, max, nm): 267, 315; +NaOH: 274, 368; +AlCl3: 274, 403; +AlCl3/HCl: 273, 403; +NaOAc: 274, 372; +H3BO3: 272, 371. 1H NMR (400 MHz, CD3OD, , ppm, J/Hz): 7.97 (2H, d, J = 8.9, H-6 , 2 ), 7.39 (1H, d, J = 15.9, H-7 ), 7.28 (2H, d, J = 8.6, H-2 , 6 ), 6.79 (2H, d, J = 8.9, H-5 , 3 ), 6.77 (2H, d, J = 8.6, H-3 , 5 ), 6.27 (1H, d, J = 1.7, H-8), 6.11 (1H, d, J = 1.7, H-6), 5.24 (1H, d, J = 7.2, Glc H-1 ), 4.32 (1H, d, J = 11.5, H-6 a), 4.19 (1H, dd, J = 12.5, 5.6, H-6 b), 3.20–4.40 (sugar protons). In addition, HPLC-UV-DAD permitted the characterization of the compound as kaempferol 3-O-[6 -O-E-p-coumaroyl]-D-glucopyranoside [8]. Compound 3. C27H30O16. UV (MeOH, max, nm): 269, 314; +NaOH: 276, 321, 365; +AlCl3: 276, 399; +AlCl3/HCl: 277, 399; +NaOAc: 275, 374; +H3BO3: 275, 372. 1H NMR (400 MHz, CD3OD, , ppm, J/Hz): 7.95 (2H, d, J = 8.9, H-6 , 2 ),

때죽나무(Styrax japonica) 수피 추출물의 항암 활성

때죽나무(Styrax japonica) 수피 메탄올 추출물에서 단리된 화합물은 TLC 및 HPLC, coulmn chromatography 방법을 이용한 실리카겔 chromatography로 분리하였다. FAB-MS, NMR 분석을 통해 화합물의 구조를 구명한 결과 분자식은 <TEX>$C_{27}H_{34}O_{11}Na$</TEX>이고 화합물명은 styraxlignolide F로 판명되었다. 인간 정상 신장세포인 HEK293을 이용한 세포독성을 살펴본 결과, 각 추출물은 <TEX>$1.0mg/m{\ell}$</TEX>의 농도에서 15.2%의 낮은 세포독성을 나타내었다. AGS와 MCF-7 세포에서의 항암활성은 <TEX>$CH_2Cl_2$</TEX> 가용부가 <TEX>$1.0mg/m{\ell}$</TEX> 농도에서 60% 이상의 억제 활성을 나타내었다. Bcl-2 단백질 정량을 통한 항암 활성 측정 결과, <TEX>$CH_2Cl_2$</TEX> 가용부가 다른 가용부 및 styraxligonlide F 보다 낮은 Bcl-2 발현량을 보였고, p53 발현량 측정에서도 <TEX>$CH_2Cl_2$</TEX> 가용부가 가장 낮은 발현량을 보였다. 인간 폐암세포(A549)를 이용한 자가사멸 측정 실험에서 <TEX>$CH_2Cl_2$</TEX> 가용부는 46.9%로 높은 사멸비율을 나타냈으며, 그다음으로 styraxlignolide F가 43.5%의 사멸비율을 나타내었다. <TEX>$CH_2Cl_2$</TEX> 분획물은 단리물질(styraxlignolide F)보다 항암 활성 효과가 높게 나타났는데 여러 가지 물질이 시너지 효과를 나타낸 것으로 사료된다. A compound has been isolated from the methanol extract of Styrax japonica bark using conventional chromatographic methods including silica gel chromatography, TLC and HPLC. The molecular formula of Styraxlignolide F analyzed by spectrometric analyses using FAB-MS, NMR was found to be <TEX>$C_{27}H_{34}O_{11}Na$</TEX>. The cytotoxicity of the styralignolide F was showed 15.2% in <TEX>$1.0mg/m{\ell}$</TEX> on human kidney cell (HEK 293). As anticancer activity of <TEX>$CH_2Cl_2$</TEX> fraction, over 60% of AGS and MCF-7 cells were inhibited in concentration of <TEX>$1.0mg/m{\ell}$</TEX>. In the results of anticancer test using quantification of Bcl-2, <TEX>$CH_2Cl_2$</TEX> fraction showed lower Bcl-2 and p53 expression than those of styraxlignolide F and other fractions. In apoptosis of human lung carcunoma cancer cell (A549), <TEX>$CH_2Cl_2$</TEX> fraction showed the highest inhibition rate (46.9%) and styralignolide F was the next (43.5%). The <TEX>$CH_2Cl_2$</TEX> fraction showed higher anti-cancer activities than isolated substance (styraxlignolide F), probably due to the crude extract showing synergic effects by other components.

Purification and Structure Elucidation of the Polyether Antibiotic Alborixin from Streptomyces pulcher CRF17

Aims: To screen Streptomyces sp. from saline soil in Pakistan, for its antimicrobial activity and to purify and identify the active metabolites produced by mass spectrometry and NMR spectroscopy. Strain identification by morphological, biochemical, physiological characterization and by 16S rRNA gene sequencing. Study Design: Cultivation in lab fermenter, solvent extraction and purification of the compounds by column chromatography, identification of the compounds by mass spectrometry and NMR spectroscopy, determination of the antimicrobial activity and cytotoxicity. Place and Duration of Study: Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan and Institute of Organic and Bio molecular Chemistry, University of Göttingen, Germany, between February, 2007 and April, 2009. Methodology: The strain was cultivated in a 20 liter fermenter (working volume 10 liters) and the culture broth was extracted with ethyl acetate, acetone and methanol. The resultant crude extract was fractionated on silica gel and the components were purified by column chromatography (silica gel, sephadex column and preparative TLC). The pure component was identified by mass spectrometry (ESI and HRESI-MS), NMR analysis (1H and 13C NMR) and by comparison with reference data, the antimicrobial activity and cytotoxicity was determined by disc diffusion assay and by micro well cytotoxicity assay against Artimia salina. Results: The morphological, biochemical and physiological characterization suggested that isolate CRF17 belongs to the genus Streptomyces. Further NCBI BLAST of the partial 16S rDNA gene sequence 1420 bp (gene bank accession number: EU294134) from the isolate CRF17 showed 99% identity and 98% query coverage towards Streptomyces pulcher. The scale up fermentation of the isolate CRF17 yielded active compound and was identified as alborixin (1). Conclusion: The isolate Streptomyces pulcher CRF17 is a potent producer of the antibiotic alborexin and can be exploited for its commercial production.

Identification of a Rare Phytosteroid from Justicia gendarussa

Justicia gendarussa L. (Acanthaceae) is an evergreen, shade-loving, highly branched shrub. Traditionally, the plant is used in the treatment of rheumatism, dysuria, fever, carbuncles, jaundice, diarrhea, pains in the head, ear, paralysis, bruises, etc. [1, 2]. Previous reports on the pharmacological studies revealed the antioxidant, antiarthritic, antiinflammatory, analgesic, antinociceptive, antifertility, anticancer, hepatoprotective, and larvicidal properties of this plant [3–5]. Numerous chemical constituents such as sterols, flavonoids, alkaloids, reducing sugars, and O-methyl ethers of benzyl alcohol were reported from J. gendarussa leaf samples [6–8]. The present study led to the discovery of a new phytosteroid in the stem as well as callus sample of J. gendarussa. The stem samples of J. gendarussa were collected from natural forests of Dakshina Kannada District, Karnataka, India and the voucher specimen (MU/AB/BN-01) was deposited at the Herbarium of Department of Applied Botany. The callus was initiated on Murashige and Skoog medium supplemented with -naphthalene acetic acid and benzyl amino purine (1.0 + 0.1 mg/L) from the sterilized stem explants, cultured in liquid medium and harvested on the 35th day [9]. The sample was dried, extracted in ether using a Soxhlet apparatus for 8–10 h, and concentrated to dryness at 40 C. The dried extract was separated in TLC (silica gel G 60 F 254, Merck, India) using acetic acid:chloroform as solvent system. The clear spot obtained on the chromatogram was separated from the silica gel, redissolved in ether, and dried. The amorphous powder obtained was used for spectroscopic analysis using UV, FTIR, 1H NMR, and LC-MS/MS. The LC-MS analysis was performed in a Thermo Finnigan Surveyor-Thermo LCQ Deca XP MAX system with a BDS Hypersil C18 column (250 mm 4.6 mm), 100% MeOH as eluent, at ambient temperature, nebulization gas helium, and electron spray ionization method with UV and PDA as detectors. The data obtained from the LC-MS/MS spectrum were compared and predicted using NIST (National Institute of Standards and Technology, Gaithersburg, MD), version 2.0a, 2002 software. Based on all the spectral data obtained, the colorless amorphous compound was predicted as a phytosteroid: 1 -carboethoxy-1 -cyano-1,2-dihydro-[1,2]cyclopropcholest-3-one (1) with an elemental composition of C32H49NO3, m/z 496 [M + H] + by ESI-MS/MS. 1 -Carboethoxy-1 -cyano-1,2-dihydro-[1,2]cycloprop-cholest-3-one (1) was isolated as a amorphous powder. The UV spectrum of the compound in ether showed absorption maxima ( max) at 282 nm, and the FTIR spectrum of the compound in KBr exhibited absorption at ( ) 1743 and 1653 cm–1 for the ester and free carbonyl groups (C=O), 2370 cm–1 for the N C group, 3450 cm–1 for -OH or -NH stretch, and 2924 cm–1 for C-H stretching. In the 1H NMR (400 MHz, MeOD, , ppm) the ethyl protons of the carboethoxy moiety appeared as a triplet and a quartet centered at 2.30 and 4.16, integrating for three and two protons, respectively. The signal due to the protons attached to the cyclopropane ring appeared as doublets at 2.05 and 2.32, integrating for one proton each. The signal due to the two methyl and the methyl groups of the isopropyl moiety overlapped with each other and appeared in the region of 0.9–1.3, integrating for 12 protons. However, the signal due to other protons overlapped with each other and appeared as multiplets in the region of 0.875–4.5. The ESI-MS/MS spectrum (ESI, 4.5 kV) showed the molecular ion peak [M + H]+ at m/z 496, consistent with the proposed molecular formula of the phytosteroid C32H49NO3. Spectroscopy was used for the detection of phytosterols in the callus tissue of Cissus quadrangularis [10], ursane type triterpenes in Olea europaea [11] and azadirachtin in cell suspension cultures of Azadirachta indica [12], triterpenoid glycosides in Justicia betonica [13], flavonoids in Punica granatum [14], and triterpenes and sesquiterpenes in Pinus densata

A New Nanostructured Stationary Phase for Ultra-Thin Layer Chromatography: A Brush-Gel Polymer Film

We report here on the fabrication and the characterization of a new stationary phase for ultrathin layer chromatography (UTLC) based on a brush-gel polymer film obtained by cross-linking poly(glycidyl methacrylate) and di(ethylene glycole)dimethacrylate. The use of polymer brushes to design ultra thin layer chromatography plates opens interesting perspectives. Very thin polymer films can be covalently grafted on a glass surface with virtually any chemical functionality. By selecting and/or chemically modifying suitable monomers, stationary phases characterized by high affinity for specific substances can be accurately tailored. The structure of the brush gel formed on glass substrates was characterized by Fourier Transform Infrared Spectroscopy (FT-IR), Atomic Force Microscopy (AFM) and Field-Emission Scanning Electron Microscopy (FESEM). The efficiency of poly(GMA-co-DEGDMA)-NH2 UTLC stationary phase compares very well with that of silica-gel TLC and other UTLC devices. However, the separation of a fluorescent dyes mixture with our UTLC device occurs faster and with less solvent compared to typical TLC methods. In addition the same plate can be re-used several times for multiple analyses without any reduction of the separation efficiency.

Enzymatic nitration of the oleic acid derivatives: Synthesis, isolation and characterization

We have synthesized the nitrated derivatives of the oleic acid based on enzymatic synthesis. Under catalysis conditions of horseradish peroxidase (HRP)/H 2 O 2 /NO, nitroalkenes are the main products. Due to the good selectivity, the reaction of HRP/H 2 O 2 /NO with unsaturated fatty acids derivative may represent a convenient and straightforward route for the small scale preparation of nitrated lipid products of potential biological interest. • We have synthesized the nitrated derivatives of the oleic acid based on chemical synthesis and enzymatic synthesis. • Under biological catalysis conditions, nitroalkenes are the main products and there are few by-products. • Due to the good selectivity, the reaction of HRP/H 2 O 2 /NO with unsaturated fatty acids derivative may represent a convenient and straightforward route for the small scale preparation of nitrated lipid products of potential biological interest. Unsaturated fatty acids are nitrated endogenously to produce nitrated lipids. Recent studies have shown that these nitrated lipids have high chemical reactivity and profound biological implications. We report an efficient, enzymatic synthesis of nitrated derivatives of the oleic acid. The methyl oleate could react with NO and horseradish peroxidase (HRP)/H 2 O 2 /NO based nitrating systems to give various nitration products which could be isolated by silica gel column and TLC fractionation, respectively. As reacting directly with NO, the obtained products contain (E)-methyl 9-nitrooctadec-9-enoate ( 1) , (E)-methyl 10-nitrooctadec-9-enoate ( 2) , (E)-methyl 9-nitrooctadec-10-enoate ( 3) and (E)-methyl 10-nitrooctadec-8-enoate ( 4) , characterized by extensive IR, NMR and GC–MS analysis. Whereas the products of the reaction between the methyl oleate and NO with the presence of HRP/H 2 O 2 were mainly composed of (E)-methyl 9-nitrooctadec-9-enoate and (E)-methyl 10-nitrooctadec-9-enoate. The improving selectivity of the products is attributed to the HRP catalysis system.

Determination of fluoxetine in the presence of photodegradation products appearing during UVA irradiation in a solid phase by chromatographic-densitometric method, kinetics and identification of photoproducts

Summary An effect of ultraviolet A (UVA) radiation on fluoxetine stability in solid phase with and without the presence of selected metal ions was studied using the chromatographic-densitometric method. Silica gel TLC F254 plates were used as the stationary phase and chloroform-methanol-ammonia 25% (45:4.5:0.5, v/v/v) as the mobile phase. The chromatograms were recorded densitometrically at a wavelength of λ = 260 nm. Both the concentration and kind of an ion have an effect on the photodegradation process. Kinetic studies demonstrated that fluoxetine photodegradation is the fastest in the presence of Cu(II) ions, decreases in the presence of Fe(III) and Fe(II) ions, and is the lowest in the presence of Al(III) ions. Under conditions established without the presence of metal ions, fluoxetine maintains photostability. The liquid chromatography-electrospray ionization/mass spectrometry (LC-ESI/MS) method was applied for the identification of decomposition products noting the presence of 3-phenyl-3-hydroxypro...

A validated stability-indicating TLC-densitometric method for the determination of stanozolol in pharmaceutical formulations

BackgroundStanozolol is a synthetic derivative of dihydrotestosterone (DHT), and one of the frequently detected anabolic steroids in doping analysis. The current work describes the development and validation of the stability-indicating TLC-densitometric method for sensitive and specific estimation of stanozolol even its degradation product being there. Precoated silica gel TLC aluminium plates were utilized as the stationary phase and the eluent comprised of petroleum ether: acetone (6:4, v/v). Densitometric analysis of stanozolol was achieved at λmax 750 nm in the absorbance mode after staining with phosphomolybdic acid (PMA). Stress degradation of stanozolol was carried out under various reaction conditions including acid, base and neutral hydrolysis, wet and dry heating treatment, oxidation, and photo-degradation. Resulted stress samples and pharmaceutical products were analyzed with the developed TLC method.ResultsThis system showed a compact spot for stanozolol at Rf value of 0.46 ± 0.02. The data of linear regression analysis indicated a good linear relationship over the range of 200–1200 ng/spot concentrations. The method was validated for robustness, precision and recovery. The LOD and LOQ were 1.6 and 5.1 ng/spot, respectively. Under various stressed conditions, stanozolol showed degradation only under acidic hydrolysis. Peak of a degraded product was well resolved from the stanazolol with reasonably different Rf value and identified as 17, 17-dimethyl-l8-nor-5α-androst-13(14)-eno [3,2c] pyrazole through 1D- and 2D-NMR spectroscopic techniques and ESI-QqTOF-MS/MS analysis.ConclusionResult reflected that the stanozolol is majorly affected by the acidic condition. Statistical analysis indicated the application of the developed stability-indicating TLC-densitometric method for routine analysis of stanozolol in the presence of its degradation product.