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Phytosterols are ubiquitous in plants, as they play an important role in cell membrane stability and as signal transducers. Over the last few decades, scientific interest in phytosterols has significantly increased. Most of the interest has focused on the cholesterol-lowering properties of phytosterols, but they may also interfere with endogenous steroid hormone synthesis. Despite this dual interest in phytosterols, accurate and fully validated methods for the quantification of phytosterols in food and feed samples are scarce. During this study an extraction and detection method for the main free phytosterols (ß-sitosterol, campesterol, stigmasterol and brassicasterol) was optimised using a fractional factorial design. Detection was carried out on a UPLC-MS/MS triple stage quadrupole apparatus. The extraction and UPLC-MS/MS detection method was fully validated according to EU Council Decision 2002/657 guidelines and Association of Analytical Chemists (AOAC) MS criteria, reaching all evaluated performance parameter requirements. The individual recoveries ranged between 95 and 104 %. Good results for repeatability and intralaboratory reproducibility (RSD %) were observed (<10 %). Excellent linearity was proven on the basis of determination coefficient (R 2 > 0.99) and lack-of-fit test (F test, alpha = 0.05). The limits of detection (LODs) and lower limits of quantification (LLOQs) in grain matrices were as low as 0.01-0.03 mg per 100 g and 0.02-0.10 mg per 100 g. This method allowed quantification of all main, free phytosterols in different grains (oats, barley, corn, malt) and it was shown that the method can be used for other solid food and feed samples as well, including new matrices such as straw, hay, mustard seeds, grass and yellow peas. Additionally, the method was shown to perform well in liquid samples low in phytosterols such as concentrate-based juices, soft drinks and beers (<5 µg per 100 mL). Graphical Abstract An extraction and detection method for the main free phytosterols (ß-sitosterol, campesterol, stigmasterol and brassicasterol) was optimised using a fractional factorial design. Detection was carried out on a UPLC-MS/MS triple stage quadrupole apparatus. The extraction and UPLC-MS/MS detection method was fully validated according to EU Council Decision 2002/657 guidelines and Association of Analytical Chemists (AOAC) MS criteria and applied on different matrices including feed and beverages.

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Careful reexamination of the published ¹H- and ¹³C-NMR spectral data of (22E)-24-methylcholesta-8(14),22-diene-3ß,5α,6ß,7α-tetraol (1), isolated from the marine-derived fungus Penicillium sp., indicates that, in reality, the compound is 5α,6α-epoxy-(22E,24R)-24-methylcholesta-8(14),22-diene-3ß,7α-diol (5).

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Three new sterols, 5α,8α-epidioxy-24-norcholesta-6,9(11),22-trien-3ß-ol (1), 5α,8α-epidioxy-cholesta-6,9(11),24-trien-3ß-ol (2), and 5α,8α-epidioxy-cholesta-6,23-dien-3ß,25-diol (3), with four known sterols (4-7) were isolated from a marine sponge Monanchora sp. Their chemical structures were elucidated by extensive spectroscopic analysis. Compounds 1 and 3-7 showed moderate cytotoxicity against several human carcinoma cell lines including renal (A-498), pancreatic (PANC-1 and MIA PaCa-2), and colorectal (HCT 116) cancer cell lines.

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A new method, reversed phase liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry (RPLC-SALDI MS) for the determination of brassicasterol (BR), cholesterol (CH), stigmasterol (ST), campesterol (CA) and ß-sitosterol (SI) in oil samples has been developed. The sample preparation consisted of alkaline saponification followed by extraction of the unsaponificable fraction with diethyl ether. The recovery of the sterols ranged from 91 to 95% with RSD less than 4%. Separation of the five major sterols on a C18 column using methanol-water gradient was achieved in about 10min. An on-line UV detector was employed for the initial sterol detection prior to effluent deposition using a laboratory-built spotter with 1:73 splitter. Off-line SALDI MS was then applied for mass determination/identification and quantification of the separated sterols. Ionization of the nonpolar analytes was achieved by silver ion cationization with silver nanoparticles used as the SALDI matrix providing limits of detection 12, 6 and 11fmol for CH, ST and SI, respectively. Because of the incorporated splitter, the effective limits of detection of the RPLC-SALDI MS analysis were 4, 3 and 4pmol (or 0.08, 0.06 and 0.08µg/mL) for CH, ST and SI, respectively. For quantification, 6-ketocholestanol (KE) was used as the internal standard. The method has been applied for the identification and quantification of sterols in olive, linseed and sunflower oil samples. The described off-line coupling of RPLC to SALDI MS represents an alternative to GC-MS for analysis of nonpolar compounds.

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BACKGROUND: Orobanche and Phelipanche species (the broomrapes) are root parasitic plants, some of which represent serious weed problems causing severe yield losses on important crops. Control strategies have largely focused on agronomic practices, resistant crop varieties and herbicides, albeit with marginal success. An alternative control method is the induction of suicidal seed germination with natural substances isolated from root exudates of host and non-host plants. RESULTS: Soyasapogenol B [olean-12-ene-3,22,24-triol(3ß,4ß,22ß)] and trans-22-dehydrocampesterol [(ergosta-5,22-dien-3-ol, (3ß,22E,24S)] were isolated from Vicia sativa root exudates. They were identified by comparing their spectroscopic and optical properties with those reported in the literature. Soyasapogenol B was very specific, stimulating the germination of O. minor seeds only, whereas trans-22-dehydrocampesterol stimulated P. aegyptiaca, O. crenata, O. foetida and O. minor. CONCLUSION: Soyasapogenol B and trans-22-deydrocampesterol were isolated for the first time from Vicia sativa root exudates, and their biological activity as stimulants of Orobanche or Phelipanche sp. seed germination was reported.

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Two new steroids isolated from EtOH extracts of the South China Sea soft coral Chromonephthea sp. were identified. One-dimensional (1D) and two-dimensional (2D) NMR experiments including COSY, HSQC, HMBC and NOESY were used for the determination of their structure.

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Four new trihydroxysteroids, sinugrandisterols A-D (1-4), have been isolated from the CH(2)Cl(2)-soluble fraction of the EtOH extract of Sinularia grandilobata. The structures of these metabolites were determined on the basis of spectroscopic (IR, MS, and 1D and 2D NMR) analysis. The cytotoxicity of 1-4 toward a limited panel of cancer cell lines is also reported.

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Four sterols were isolated from a brown alga endophytic fungus NO. ZZF36 from the South China Sea. Their structures were identified as brassicaterol(2), ergosterol(2), ergosterol peroxide(3),7,22(E)-ergostadiene-beta,5alpha,6beta-triol(4) by spectroscopic methods.

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Activity guided fractionations led to the isolation of two antitumor compounds 5 alpha,8 alpha-epidioxy-24(R)-methylcholesta-6,22-dien-3 beta-D-glucopyranoside and 5,6-epoxy-24(R)-methylcholesta-7,22-dien-3 beta-ol from the methanol extract of Cordyceps sinensis. Two previously known compounds, ergosteryl-3-O-beta-D-glucopyranoside and 22-dihydroergosteryl-3-O-beta-D-glucopyranoside were also isolated. The structures of hitherto unknown sterols were established by 1D and 2D NMR spectroscopic techniques with the former synthesized in order to confirm the identity of the sugar moiety by chemical correlation. The glycosylated form of ergosterol peroxide was found to be a greater inhibitor to the proliferation of K562, Jurkat, WM-1341, HL-60 and RPMI-8226 tumor cell lines by 10 to 40% at 10 micrograms/ml than its previously identified aglycone, 5 alpha,8 alpha-epidioxy-24(R)-methylcholesta-6,22-dien-3 beta-ol.

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We report that silver ion HPLC provides remarkable separations of C27 sterols differing only in the number or location of olefinic double bonds. This technique has been extended to LC-MS, analysis of purified components by GC, GC-MS, and 1H NMR, and to its use on a semipreparative scale. The application of this methodology for the demonstration of the catalysis, by rat liver microsomes, of the conversion of 7-dehydrocholesterol to cholesta-5,8-dien-3 beta-ol is also presented.

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A number of neutral marine steroids such as desmosterol, campesterol, brassicasterol, gorgosterol, and other trace steroids were isolated from the coelomic fluid of ripe Nereis succinea and checked for biological activity as sex pheromones on swarming specimens of Platynereis dumerilii and Nereis succinea. No significant influence of synthetic gorgosterol or a natural extract of gorgosterol or the other identified steroids on the swarming behavior was observed.

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Six new delta 8- and delta 8(14)-5 alpha,6 alpha-epoxysterols (1-6) have been isolated from a collection of the Mediterranean sponge Spongia officinalis. The structures of the new metabolites, isolated as diacetate derivatives, have been determined by spectral analyses and an x-ray diffraction experiment performed on the diacetyl derivative of 1.

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Four new polyoxygenated sterols 2-5 were isolated from the marine black coral Antipathes subpinnata. The structures of these compounds, including stereochemical details, were deduced by ms, 1H- and 13C-nmr, 1H-1H COSY, and nOe difference spectroscopy. Compounds 2-5 are lethal to brine shrimp (Artemia salina).

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An examination of the Australian sponge Phakellia aruensis led to the isolation and identification of sterols with six different nuclei. Eight new sterols were isolated which included three delta 15-A-nor sterols, three delta 7-A-nor sterols, and two saturated A-nor sterols. Their structures were established through mass spectrometry and 1H-NMR spectral studies.

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Reinvestigation of Cynoglossum creticum led to the isolation of the previously reported echinatine [1] and heliosupine [2] as well as rinderine [3], 7-angelylheliotridine [4] and a new alkaloid, cynoglossamine [5]. The structures have been determined by spectral means (ir, ms, 1H-13C HETCOR nmr), comparison with literature data and authentic samples, and/or syntheses. In addition, 1 and all three of its isomers 3, 6, and 7 and other semisynthetic analogues (8-13) were prepared and characterized.

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A minor C27 sterol, glaucasterol, was isolated from the soft coral Sarcophyton glaucum. Based on the spectroscopic evidence and the correlation to cholestanol and 26-nor-27-homocholestanol, its structure was proposed to be 24 epsilon,25 epsilon-24,26-cyclocholesta-5,22E-dien-3 eta-ol (1), the first example of a natural C27 sterol having a cyclopropane ring in the side chain.

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A relatively simple method is described for the isolation of zymosterol (5 alpha-cholesta-8, 24-dien-3 beta-ol) of high purity from baker's yeast. Also presented are detailed spectral properties, including 13C NMR spectral analyses, of zymosterol and its acetate derivative.

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The sterol mixture of the southern Japan's soft coral, Sarcophyton glaucum, was found to contain 11 sterols including a novel sterol, 23,24 xi-dimethylcholesta-5,22-dien-3 beta-ol and a new diunsaturated C29 sterol. 22,23-Dihydrobrassicasterol and gorgosterol were the major components in free- and esterified sterols respectively. Brassicasterol was found in S. glaucum, in contrast to the ubiquity of 24-epibrassicasterol in the marine invertebrates in the northern districts. The new sterol (sarcosterol) was isolated; its structure as 23 xi, 24 xi-dimethylcholesta-5, 17(20)-trans-dien-3 beta-ol was based on spectra evidence and comparison with cholesta-5, 17(20)-trans-dien-3 beta-ol.

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A method of isolating pure fractions of 4 alpha-methyl-5 alpha-ergosta-8,24(28)-dien-38-ol for sterol intermediate studies is described. Starvation cultures of Neurospora crassa readily incorporate exogenous mevalonic acid into the sterol ester fraction. Isolation involves a simple solvent extraction and two chromatograms. Only the ester fraction yielded the required purity. Radioactive 4 alpha-methyl-5 alpha-ergosta-8,24(28)-dien-3 beta-ol is readily produced from DL-[2-14C] mevalonic acid.

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