Oligolignans in the wood of <Emphasis Type="Italic">Picea obovata</Emphasis> Ledeb.

We studied the chemical composition of the phenolic complex and the structure of oligomeric lignans in Siberian spruce (Picea obovata Ledeb.). We used the wood of Siberian spruce collected near the city of Irkutsk. The extractives were isolated from ground wood (particle size: 10–15 mm; humidity: 5.9%) by three-stage acetone extraction. The extract was separated by consecutive treatment with the solvents with increasing polarity: hexane, ethyl acetate and n-butanol. The main amount of phenolic compounds (lignans) was concentrated in the ethyl acetate fraction and it was 0.7% in absolutely dry wood (adw).The ethyl acetate fraction of spruce wood extract was separated by silica gel column; a chloroform-acetone mixture was used as the eluent (the concentration of acetone in the mixture was increased from 0 to 100%). Monomeric lignans (~60–65% of the ethyl acetate fraction of spruce wood extract), oligomeric lignans (~20–25%), and polymer lignans (~12–15%) were isolated.We also obtained ¹³C nuclear magnetic resonance (NMR) spectra for the main monomeric, oligomeric and polymeric lignan compounds of phenolic complexes. It was found that oligomeric and polymeric fractions contain monomeric lignan units with the butyrolactone cycle, primarily, the fragments with hydroxymatairesinol structure. Oligomeric lignans contain the fragments with a pinoresinol and lariciresinol structure. All the monomeric structural units are characterized by the guaiacyl substitution type of the aromatic cycles.A preliminary study has been performed on the antiviral and antioxidant activity of the ethyl acetate fraction of acetone extract of Siberian spruce wood. It was found that the lignan complex is active against Coxsackie B4 virus in cell culture and in the pancreatitis model in white mice, reducing the activity of enteroviruses in the cell culture approximately 100 times. The antioxidant activity rate of polyphenol complex of Siberian spruce wood is comparable to that of a known antioxidant dihydroquercetin.     

Red Mexican grapefruit: a novel source for bioactive limonoids and their antioxidant activity

Citrus limonoids have shown to inhibit the growth of cancer in colon, lung, mouth, stomach and breast in animal and cell culture studies. For the first time in the present study, an attempt has been made to isolate antioxidant fractions and five limonoids from red Mexican grapefruit seeds. Defatted seed powder was successively extracted with hexane, ethyl acetate (EtOAc), acetone, methanol (MeOH) and MeOH/water and the extracts were concentrated under vacuum. Radical scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and total phenolic content were also measured for comparison with the antioxidant capacity in the phosphomolybdenum method for the above extracts. Acetone and MeOH extracts, respectively, showed the highest (85.7%) and lowest (53.3%) radical scavenging activity, at 500 ppm. The total phenolic contents were found to be highest in the acetone extract (15.94%) followed by the MeOH extract (5.92%), ethyl acetate extract (5.54%) and water extract (5.26%). Antioxidant capacity of the extracts as equivalents to ascorbic acid (micromol/g of the extract) was in the order, EtOAc extract > acetone extract > water extract > methanol extract. Furthermore, the EtOAC and acetone extracts were loaded onto silica gel columns to obtain four limonoid aglycons. MeOH fraction was loaded onto a dowex-50 and sepabeads resin column to obtain a limonoid glucoside. The purity of the isolated five compounds was analyzed by HPLC using a C18 column and UV detection at 210 nm. Finally, the structures of the compounds were identified as obacunone, nomilin, limonin, deacetylnomilin (DAN) and limonin-17-beta-D-glucopyranoside (LG) using 1H and 13C NMR studies.     

Effect of biomass pre-treatment and solvent extraction on beta-carotene and lycopene recovery from Blakeslea trispora cells

The production of carotenoids from Blakeslea trispora cells in a synthetic medium has been reported, with the main products being beta-carotene, lycopene, and gamma-carotene. The effect of biomass pretreatment and solvent extraction on their selective recovery is reported here. Eight solvents of class II and III of the International Conference of Harmonization: ethanol, methanol, acetone, 2-propanol, pentane, hexane, ethyl acetate, and ethyl ether, and HPLC analysis were used for the evaluation of their selectivities towards the three main carotenoids with regard to different biomass pre-treatment. The average C(max) values (maximum concentration of caronoids in a specific solvent) were estimated to 16 mg/L with the five out of eight solvents investigated, whereas methanol, pentane, and hexane gave lower values of 10, 11, and 9 mg/L, respectively. The highest carotenoid yield was obtained in the case of wet biomass, where 44-56% is recovered with one solvent and three extractions and the rest is recovered only after subsequent treatment with acetone; thus, four extractions of 2.5 h are needed. Two extractions of 54 min are enough to recover carotenoids from dehydrated biomass, with the disadvantage of a high degree of degradation. Our results showed that, for maximum carotenoid recovery, ethyl ether, 2-propanol, and ethanol could be successfully used with biomass without prior treatment, whereas fractions enriched in beta-carotene or lycopene can be obtained by extraction with the proper solvent, thus avoiding degradation due to time-consuming processes.     

Purification of Xylitol from Fermented Hemicellulosic Hydrolyzate Using Liquid–Liquid Extraction and Precipitation Techniques

Xylitol was produced by Candida guilliermondii by fermentation of sugarcane bagasse hemicellulosic hydrolysate. Undesirable impurities were extracted from the broth using either ethyl acetate, chloroform or dichloromethane. The best results on clarification of the broth without xylitol loss were obtained with ethyl acetate. When ethanol, acetone or tetrahydrofuran were used for precipitation of impurities, only tetrahydrofuran clarified the fermented broth, but a high xylitol loss (~30%) was observed.     

Effect of Biomass Pre-Treatment and Solvent Extraction on β-Carotene and Lycopene Recovery from Blakeslea trispora Cells

Abstract

The production of carotenoids from Blakeslea trispora cells in a synthetic medium has been reported, with the main products being β-carotene, lycopene, and γ-carotene. The effect of biomass pretreatment and solvent extraction on their selective recovery is reported here. Eight solvents of class II and III of the International Conference of Harmonization: ethanol, methanol, acetone, 2-propanol, pentane, hexane, ethyl acetate, and ethyl ether, and HPLC analysis were used for the evaluation of their selectivities towards the three main carotenoids with regard to different biomass pre-treatment. The average C_max values (maximum concentration of caronoids in a specific solvent) were estimated to 16 mg/L with the five out of eight solvents investigated, whereas methanol, pentane, and hexane gave lower values of 10, 11, and 9 mg/L, respectively. The highest carotenoid yield was obtained in the case of wet biomass, where 44–56% is recovered with one solvent and three extractions and the rest is recovered only after subsequent treatment with acetone; thus, four extractions of 2.5 h are needed. Two extractions of 54 min are enough to recover carotenoids from dehydrated biomass, with the disadvantage of a high degree of degradation. Our results showed that, for maximum carotenoid recovery, ethyl ether, 2-propanol, and ethanol could be successfully used with biomass without prior treatment, whereas fractions enriched in β-carotene or lycopene can be obtained by extraction with the proper solvent, thus avoiding degradation due to time-consuming processes.     

Quantitative determination of phyllanthin and hypophyllanthin in Phyllanthus species by high-performance thin layer chromatography

A simple, precise and rapid high-performance thin-layer chromatographic method has been developed for the estimation of phyllanthin (1) and hypophyllanthin (2), the important lignans of Phyllanthus species, especially Phyllanthus amarus. Separation of 1 and 2 was carried out on silica gel 60 F254 layers eluted with hexane:acetone:ethyl acetate (74:12:8), and the analytes were visualised through colour development with vanillin in concentrated sulphuric acid and ethanol. Scanning and quantification of spots was performed at 580 nm. Recoveries of 1 and 2 were 98.7 and 97.3%, respectively. The method was validated and the peak purities and limits of detection and quantification were determined.     

Purification of Antibiotics from Physarum Gyrosum by High Pressure Liquid Chromatography

A family of five antibiotic substances was isolated from the slime mold Physarum gyrosurn by high pressure liquid chromatography (HPLC). For this purpose, mold was cultured for two weeks in a liquid medium. Soluble products were harvested by rotary evaporation of medium and extraction with 1-butanol. Paper chromatography in ethyl acetate :pyridine:water (2:2:1 v/v) was used for preliminary fractionation. Active components were separated by HPLC with a reverse-phase column packed with Bondapack C₁₈/Porasil B (Waters Associates) and were eluted with a linear gradient of methanol:water increasing from 70 to 100% methanol over 90 minutes. Puri-fication was completed by rechromatographing individual fractions. Purity of the active components was verified by HPLC and thin layer chromatography. Activity assays against Bacillus cereus showed these materials to be bacteriostatic rather than bacteriocidal.     

Measurement of F2-isoprostanes, hydroxyeicosatetraenoic products, and oxysterols from a single plasma sample

Oxidized lipids such as F2-isoprostanes (F2-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), and cholesterol oxidation products (COPs) are widely believed to be involved in multiple diseases. Usually, each product is measured individually in separate blood samples. In this study we describe a method allowing us to measure F2-IsoPs, HETEs, COPs, and arachidonate using a single sample. Plasma (1 ml) samples from healthy volunteers were diluted with heavy isotopic standards, hydrolyzed in alkali with organic solvent, and then subjected to anionic-exchange solid-phase extraction (SPE). After the SPE column was washed, hexane and hexane/ethyl acetate portions were collected and combined for COPs measurement. Thereafter the column was loaded with hexane/ethanol/acetic acid and fractions were collected for total F2-IsoPs, total HETEs, and arachidonate measurement. All compounds in the eluates were measured by gas chromatography-mass spectrometry. The efficiency of SPE and reproducibility for all compounds measured were high. Levels of total F2-IsoPs (0.45+/-0.26 ng/ml (n=157)), total HETEs (34.06+/-16.35 ng/ml (n=21)), total arachidonate (68.36+/-24.45 microg/ml (n=33)), and COPs (7-ketocholesterol, 12.25+/-6.56 ng/ml; 7beta-hydroxycholesterol, 6.32+/-3.46 ng/ml; 7alpha-hydroxycholesterol, 15.06+/-7.06 ng/ml; 24-hydroxycholesterol, 41.39+/-18.22 ng/ml; and 27-hydroxycholesterol, 29.08+/-16.79 ng/ml (n=26)) were recorded in healthy subjects (age range 20 to 66 years; average male to female ratio 1:1).     

Antimycobacterial activity of two natural alkaloids,vasicine acetate and 2-acetyl benzylamine,isolated from Indian shrub <Emphasis Type="Italic">Adhatoda vasica</Emphasis> Ness. leaves

In folk medicine, Adhatoda vasica Ness. (Acanthaceae) is used to treat asthma and cough. The leaves of A.vasica were powdered and extracted with hexane, ethyl acetate and methanol. The hexane extract showed 97% reduction in colony-forming units (CFU) at 100 μg/ml. The hexane extract was subjected to column chromatography. Two natural compounds, vasicine acetate and 2-acetyl benzylamine, were isolated from it. They were bioassayed against Mycobacterium tuberculosis. The two compounds showed strong antimycobacterial activity. Vasicine acetate and 2-acetyl benzylamine isolated from hexane extract of A.vasica leaves, significantly inhibited M. tuberculosis and one multi-drug-resistant (MDR) strain and one sensitive strain at 200 and 50 μg/ml, respectively. Our study demonstrated that both the compounds, vasicine acetate and 2-acetyl benzylamine, could be evaluated further for developing a drug to control M. tuberculosis.     

Quantitative analysis of plasma neutral glycosphingolipids by high performance liquid chromatography of their perbenzoyl derivatives.

A quantitative high performance liquid chromatography method for the analysis of neutral glycosylceramides as their perbenzoyl derivatives has been devised. Samples containing more than 2.5 nmol each of mono-, di-, tri-, and tetraglycosylceramide are benzoylated with 10% benzoyl chloride in pyridine at 37degrees C for 16 hr. The products are separated from excess reagents by solvent distribution and injected onto a pellicllar silica gel (Zipax) column (2.1 mm X 50 cm). The derivatives are eluted with a 10 min linear gradient of 2-17% ethyl acetate in hexane at 2 ml/min and absorbance at 280 nm is recorded. The detector response was proportional to the weight of sample used (2-30 nmol) and the lower limit of detection was about 70 pmol. The procedure has been applied to the quantitative analysis of erythrocyte and plasma glycolipids. As little as 0.5 ml of plasma can be used for analysis. The relative standard deviation of repetitive analyses ranged between 2.0% for glucosylceramide to 5.4% for galactosyllactosylceramide.     

Purification of fatty acid ethyl esters by solid-phase extraction and high-performance liquid chromatography

We have developed a two-step method to purify fatty acid ethyl esters (FAEE) using solid-phase extraction (SPE), with a recovery of 70±3% (mean±S.E.M.) as assessed using ethyl oleate as a recovery marker from a standard lipid mixture in hexane. The first step of the SPE procedure involves application of a lipid mixture to an aminopropyl-silica column with simultaneous elution of FAEE and cholesteryl esters from the column with hexane. Gas chromatographic analysis of FAEE without interference from cholesteryl esters may be performed using the eluate from the aminopropyl-silica column, thus eliminating the need for an octadecylsily (ODS) column in this case. The FAEE can then be separated from the cholesteryl esters, if necessary, by chromatography on an ODS column and elution with isopropanol-water (5:1, v/v). Both the aminopropyl-silica and ODS columns were found to be effective for up to four uses. To permit isolation of specific FAEE species following isolation of total FAEE by the two-step SPE method, we have also developed a purification scheme for individaal FAEE by high-performance liquid chromatography (HPLC). Thus, this simple method allows for reproducible isolation of total FAEE by SPE and isolation of individual FAEE species by HPLC.     

Determination of the geometrical diarylpropenamine isomers in feces by high-performance liquid chromatography

Diarylpropenamine derivatives are a class of compounds which have been evaluated as potential drug candidates. Here a specific and reproducible HPLC method for the determination of cis- and trans-isomers of the unsubstituted derivative, 3-(4′-bromo-[1,1′-biphenyl]-4-yl)-3-(4-X-phenyl-N,N-dimethyl-2-propen-1-amine (I, where X=H) in feces is described. The analyte I and internal standard, nitro derivative (II, where X=NO₂), were isolated from the basified biological matrix using a liquid–liquid extraction with ethyl acetate followed by a solid-phase procedure performed on a silica cartridge. The organic phase was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The analytes were eluted with ethyl acetate–hexane–triethylamine (59:40:1) in HPLC column (silica) and detected by UV spectrophotometry at 272 nm. Linearity, precision and accuracy data for feces standards after extraction were acceptable. The method has been applied to analyses of feces samples from rats dosed with I, in which it could be anticipated that fecal excretion is quantitatively the major route for I elimination.     

Simultaneous gas chromatographic determination of methamphetamine, amphetamine and their p-hydroxylated metabolites in plasma and urine

We report a method for the simultaneous determination of methamphetamine, amphetamine and their hydroxylated metabolites in plasma and urine samples using a GC-NPD system. The analytical procedures are: (1) adjust the sample to pH 11.5 with bicarbonate buffer, saturate with NaCl and extract with acetate; (2) back-extract the amines in the ethyl acetate fraction with 0.1 M HCl; (3) adjust the pH of the acid fraction to 11.5 and follow by extraction in ethyl acetate; (4) reduce the volume of ethyl acetate under nitrogen and derivatize the concentrate with trifluoroacetic anhydride or heptaflourobutyric anhydride before the GC analysis. The derivatives were separated on a GC-NPD system equipped with a HP-5 column of 25 m×0.32 m I.D. and a 0.52 μm film of 5% phenylmethylsilicone. The detection limit (taking a signal-to-noise ratio of 2) of heptafluorobutyl derivatives of methamphetamine and its metabolites in plasma and the trifluoroacetyl derivatives in urine was 1 ng/ml (22 pg on column). The limit of quantitation of the heptafluorobutyl derivatives in the plasma was 1 ng/ml (22 pg on column), and that of the trifluoroacetyl derivatives in urine was 20 ng/ml (73 pg on column). The between-day variation was from 0.9 to 17.4% and within-day variation from 0.9 to 8.3%. This method was used successfully in the quantitative determination of methamphetamine and its p-hydroxylated metabolites in the plasma and urine of human subjects.     

Semiochemicals from ovaries of gravid females attract ovipositing female houseflies,Musca domestica

Chemical signals originating from the ovaries of gravid females of Musca domestica (Diptera: Cyclorrhapha: Muscidae) attract ovipositing females to common egg-laying sites. Behavioral experiments indicated that females preferred to oviposit in fermented wheat bran containing ovaries from reproductively mature houseflies. Females preferred to oviposit in fermented wheat bran than wet wheat bran. This effect was additive with the attraction to housefly ovaries. Solvent extracts from housefly ovaries were attractive to gravid females. Extracts obtained with hexane were most attractive to gravid females for egg laying, and extracts obtained with ethyl acetate attracted more egg laying than extracts obtained by dichloromethane. Gas chromatography (GC) and gas chromatography coupled with mass spectrometry (GC-MS) analysis showed that tricosane and (Z)-9-tricosene were the main components of the hexane extracts. Both tricosane and (Z)-9-tricosene were shown to elicit dose-dependent aggregation of gravid females in oviposition bioassays, but high doses of either chemical were not attractive.     

Preparation of 4,15-diacetoxyscirpenol from cultures of Fusarium sambucinum NRRL 13495.

Filtrates of Fusarium sambucinum NRRL 13495 grown in a stagnant culture for 9 days contained up to 458 +/- 60 (mean +/- standard error; n = 3) mg of 4,15-diacetoxyscirpenol per liter depending on culture conditions. Extraction with ethyl acetate, chromatography on a column of silica gel, and crystallization from mixtures of ethyl acetate and hexane provided pure material in 96% yield.     

Preparation of 4,15-diacetoxyscirpenol from cultures of Fusarium sambucinum NRRL 13495

Filtrates of Fusarium sambucinum NRRL 13495 grown in a stagnant culture for 9 days contained up to 458 +/- 60 (mean +/- standard error; n = 3) mg of 4,15-diacetoxyscirpenol per liter depending on culture conditions. Extraction with ethyl acetate, chromatography on a column of silica gel, and crystallization from mixtures of ethyl acetate and hexane provided pure material in 96% yield.     

益智有效抗氧化成分的分离条件的研究

探讨了干柱层析法分离益智渣及益智乙醇提取物的抗氧化成分。实验中采用乙酸乙酯/环己烷(3∶7)、氯仿/甲醇(9∶1)为展开剂依次二次干柱层析分离出抗氧化物质。结果表明,益智渣及益智乙醇提取物均有较强的抗氧化性,益智渣的H2O2清除能力强于益智乙醇提取物。     

Inhibitory effect of Terminalia chebula Retz. fruit extracts on digestive enzyme related to diabetes and oxidative stress

Terminalia chebula fruit extracts were prepared sequentially with hexane, ethyl acetate, methanol and methanol–water (70:30) and tested for their α-glucosidase inhibitory and antioxidant potential. The study resulted in the formulation of an extract with high α-glucosidase inhibitory potential (IC₅₀ 0.19?±?0.03 µg mL^?1) enriched with hydrolysable tannins. Also, each of the extract was chemically characterized by reversed-phase high-performance liquid chromatography on the basis of their marker compounds chebulagic acid, chebulinic acid and corilagin in order to give explanation to the significant activity shown by the extracts. The antioxidant potential of the highly active extract was evaluated in the cellular level also using superoxide dismutase, glutathione S-transferase and induced oxidative stress assays. The results indicated the possibility of using the extract as a nutraceutical health supplement in the management of type 2 diabetes.     

Isolation of Fucoxanthin from the Rhizoid of Laminaria japonica Aresch

Fucoxanthin was extracted from the intact rhizoid of Laminariajaponica Aresch with dimethyl sulfoxide （DMSO）, and then recovered from the DMSO extract by partitioning into ethyl acetate and subsequent evaporation. Some isolation conditions such as solvent volume and extraction time were screened. The quantity and quality of the extracted fucoxanthin were determined by spectral analysis （absorption spectra and fluorescence emission spectra）. The results indicated that： （1） the average total content of fucoxanthin was 122.1μg in 1 g of fresh L. japonica rhizoid; （2） in comparison with the widely used organic solvent, acetone, DMSO was much more effective for the extraction of fucoxanthin; （3） both DMSO volume and extraction time influenced extraction efficiency such as the recovery rate and purity of fucoxanthin （1 g of fresh L. japonica rhizoid treated with 4 mL DMSO for 60 min, yielded 〉 88% of the total fucoxanthin with purity 0.63）; （4） when （NH4）2SO4 concentration was in the range of 0.5-1.0 mol/L, the pigments rapidly and entirely moved from DMSO into the ethyl acetate phase; （5） the ethyl acetate and DMSO were recycled using a rotary evaporator.     

The quantification of erlotinib (OSI-774) and OSI-420 in human plasma by liquid chromatography-tandem mass spectrometry

An accurate and precise method was developed using HPLC-MS/MS to quantify erlotinib (OSI-774) and its O-desmethyl metabolite, OSI-420, in plasma. The advantages of this method include the use of a small sample volume, liquid-liquid extraction with high extraction efficiency and short chromatographic run times. The analytes were extracted from 100 microL plasma volume using hexane:ethyl acetate after midazolam was added to the sample for internal standardization. The compounds were separated on a Phenomenex C-18 Luna analytical column with acetonitrile:5 mM ammonium acetate as the mobile phase. All compounds were monitored by tandem mass spectrometry with electrospray positive ionization. The intra-day accuracy and precision (% coefficient of variation, % CV) estimates for erlotinib at 10 ng/mL were 90% and 9%, respectively. The intra-day accuracy and precision estimates for OSI-420 at 5 ng/mL were 80% and 4%, respectively. This method was used to quantify erlotinib and OSI-420 in plasma of patients (n=21) administered 150 mg erlotinib per day for non-small cell lung cancer.