Untersuchungen zur Anreicherung von Ubichinon-9 aus Lipid-Kohlenwasserstoff-Fraktionen mittels Kurzwegdestillation

Short path distillation was used for the enrichment of ubiquinone-9 from a lipid hydrocarbon extract. The main components of the lipid hydrocarbon extract were hydrocarbons of gas oil (b. p. 513 to 653 K), phosphatides, glycerides, and fatty acids. Phosphatides were isolated by extraction with acetone before distillation. The acetone soluble fraction was distilled by a pressure of ≦ 0.5 kPa and a temperature of 433 to 453 K. The concentration of the ubiquinone-9 in the residue of distillation was three times higher than in the acetone soluble fraction. Ubiquinone-9 can be obtained by a simplified separation from the enriched fraction.     

Lipase catalyzed interesterification of palm stearin and rice bran oil blends for preparation of zero trans shortening with bioactive phytochemicals

Palm stearin (PS) and rice bran oil (RBO) blends of varying proportions were subjected to enzymatic interesterification (EIE) using a 1,3 specific immobilized lipase. The interesterified blends were evaluated for their physicochemical characteristics and bioactive phytochemical contents using differential scanning calorimeter (DSC), X-ray diffraction (XRD), gas chromatography (GC) and high performance liquid chromatography (HPLC). The blends of PS and RBO in different proportions (40:60, 50:50, 60:40 and 70:30) had saturated fatty acid content and unsaturated fatty acid content in the range of 37.6-52.0% and 48.0-62.4%, respectively. The blends 40:60, 50:50 and 60:40 showed a considerable reduction in their highest melt peak temperature (TP) and solid fat content (SFC) on EIE. The interesterified blends retained bioactive phytochemicals like tocols (839-1172 ppm), sterols (4318-9647 ppm), oryzanols (3000-6800ppm) and carotene (121-180 ppm). XRD studies demonstrated that the interesterified blends contained beta and beta' polymorphic forms.     

Antiproliferative activity is predominantly associated with ellagitannins in raspberry extracts

Raspberry extracts enriched in polyphenols, but devoid of organic acids, sugars and vitamin C, were prepared by sorption to C18 solid phase extraction matrices and tested for their ability to inhibit the proliferation of human cervical cancer (HeLa) cells in vitro. The raspberry extract reduced proliferation in a dose-dependent manner whether this was judged by cell number or measurements of cell viability. However, measurements based on cell viability were more accurate and gave an EC(50) value of 17.5 microg/ml gallic acid equivalents (GAE) at day 4 of culture. Raspberry extracts were fractionated by sorption to Sephadex LH-20 into an unbound fraction, which was obviously enriched in anthocyanins, and a bound fraction. The unbound anthocyanin-enriched fraction was much less effective in reducing proliferation then the original extract and gave an EC(50) value estimated at 67 microg/ml. The LH-20 bound fraction was more effective than the original raspberry extract (EC(50)=13 microg/ml) suggesting that the main anti-proliferative agents were retained in the bound fraction. Analysis of the original extract, the unbound and the LH20 bound fractions by LC-MS confirmed that the unbound fraction was enriched in anthocyanins and the bound fraction primarily contained ellagitannins. The ellagitannin-rich bound fraction had the highest antioxidant capacity as measured by the ferric reducing antioxidant potential (FRAP) assay. The mechanism by which the ellagitannins inhibit proliferation of cancer cells is discussed.     

Effect of ferulic acid from Hibiscus mutabilis on filarial parasite Setaria cervi: Molecular and biochemical approaches

In the reported work the in vitro activity of a methanolic extract of leaves of Hibiscus mutabilis (Malvaceae) against bovine Setaria cervi worms has been investigated. Bioassay-guided fractionation led to isolation of ferulic acid from ethyl acetate fraction. The crude extract and ferulic acid, the active molecule, showed significant microfilaricidal as well as macrofilaricidal activities against the microfilaria (L(1)) and adult of S. cervi by both a worm motility and MTT reduction assay. The findings thus provide a new lead for development of a filaricidal drug from natural products. To examine the possible mechanism of action of ferulic acid, the involvement of apoptosis in adult worms of S. cervi was investigated. We found extreme cellular disturbances in ferulic acid-treated adult worms characterized by chromatin condensation, in situ DNA fragmentation and nucleosomal DNA laddering. In this work we are reporting for the first time that ferulic acid exerts its antifilarial effect through induction of apoptosis and by downregulating and altering the level of some key antioxidants (GSH, GST and SOD) of the filarial nematode S. cervi. Our results have provided experimental evidence supporting that ferulic acid causes an increased proapoptotic gene expression and decreased expression of anti-apoptotic genes simultaneously with an elevated level of ROS and gradual dose dependent decline of parasitic GSH level. We also observed a gradual dose dependent elevation of GST and SOD activity in the ferulic acid treated worms.     

The effects of insulin,cycloheximide and phalloidin on the content of actin and p35 in extracts prepared from the nuclear fraction of Krebs II ascites cells

The nuclear fraction isolated from Krebs II ascites cells following cell disruption by nitrogen cavitation was separated into four fractions by salt/detergent extraction: NP-40 soluble fraction, 130 mM KCl extract, DOC/Triton × 100 soluble fraction and salt/detergent treated nuclei. The protein composition of the individual fractions was studied by SDS-PAGE and the relative amounts of actin and a 35 kDa protein (p35) were measured from gel scans. There was a time-dependent shift of actin from the 130 mM KCl extract to the NP-40 soluble fraction upon storage of the nuclear fraction on ice, indicating a progressive depolymerization of microfilaments. Compared with actin there was a slower release of p35 into the NP-40 soluble fraction. The results suggest that p35 is not integrated in the microfilament network. Phalloidin, which stabilizes the microfilaments, enriched the amount of both proteins in the 130 mM KCl extracts, together with a series of other proteins in the range 50–205 kDa. The presence of phalloidin also resulted in a large increase in the actin content in both the DOC/Triton × 100 extract and the fraction containing salt/detergent treated nuclei. Incubation of cells with insulin and/or cycloheximide enriched the amount of actin in the 130 mM KCl fraction. The results show that short term incubation of cells with phalloidin, insulin or cycloheximide increases the actin content of the nuclear fraction and also affects the presence of several other proteins.     

Supercritical CO2 extraction of lipids from grain sorghum dried distillers grains with solubles

Experiments were carried out on a lab supercritical CO(2) extraction system to determine the effects of extraction conditions, including mass ratio of CO(2) consumed to distillers dry grain with solubles (DDGS) extracted, extraction pressure, extraction temperature and time, on yield and composition of extracted lipids. A maximum lipid yield of 150 g/kg DDGS was achieved with a mass ratio approximately 45, an extraction pressure at 27.5 MPa, an extraction temperature at 70 degrees C and an extraction time of 4 h. Under these extraction conditions, the contents of tocols, phytosterols, policosanols and free fatty acids were 0.44, 15.6, 31.2 and 155.3 mg/g in the extract. Experimental results indicated that shorter extraction time and higher flow rate of CO(2) can achieve higher contents of tocols, phytosterols and policosanols but lower content of free fatty acids in the lipid extract. Extraction conditions had no observed effects on the composition of free fatty acids in the extract. Palmitic, oleic and linoleic acids were three main free fatty acids extracted and constituted about 94% of all free fatty acids.     

Induction of Chlorosis,ROS Generation and Cell Death by a Toxin Isolated from Pyricularia oryzae

The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.     

Rapid preparation of samples for compositional sugar analysis of the "degraded polysaccharide" fraction of lipopolysaccharides from Vibrio cholerae

A simple and rapid method was devised for direct isolation and fractionation of the "degraded polysaccharide" (DPS) fraction of O-antigenic (or endotoxic) lipopolysaccharides (LPS) directly from heat-killed Vibrio cholerae (O1 and non-O1) cells without separating the LPS. Neither phenol-water extraction nor ultracentrifuge is needed in this method. V. cholerae NIH 41 was used as standard. The cells (3-5 g wet weight) were heated in 5% acetic acid at 100 C for 1.5 hr. The acetic acid extract obtained as the supernatant by centrifugation was evaporated to dryness in vacuo, and the resultant residue was dissolved in 10 ml of distilled water. The solution was mixed with 2 volumes of acetone, and the supernatant obtained by centrifugation was mixed with 5 volumes of acetone and centrifuged. Fraction Sed. II was recovered as the precipitate, while the supernatant was evaporated to dryness in vacuo, yielding fraction Sup. III. Sed. II had a sugar composition that was identical, at least qualitatively, to that of DPS isolated from LPS of the corresponding strain except for the absence of a fructose component in the case of V. cholerae NIH 41, while instead Sup. III from V. cholerae NIH 41 contained fructose.     

Effect of stage of development on chemical yields in smooth sumac,Rhus glabra L.

Smooth sumac (Rhus glabra L.) has been identified as a potential source of polyphenols, oil, and polymeric hydrocarbon for the chemical industry. The effect of stage of development (prebud, full bud, full flower and seed set) on acetone extract (‘polyphenols’ and ‘oil’) and hexane extract (polymeric hydrocarbon) yields was studied in two Beltsville, Maryland (USA) populations in 1982 and 1983. Most extract was found in the leaves, but a substantial amount was also present in stem tissue. Leaf fraction acetone extract yields were generally highest at the full-flower stage and stem yields were generally highest at the seedset stage. Leaf fraction hexane extract yield was highest at the seed-set stage whereas stage of development had no consistent effect on stem fraction yields. For Population 2, there was a fairly strong inverse relationship between yields of leaf fraction acetone and leaf fraction hexane extract both years, and, for Population 1, a fairly strong direct relationship between yields of stem fraction acetone extract and stem fraction hexane extract in 1982. Data indicate that harvesting at the full-flower stage would maximize acetone extract yields, whereas harvesting at the seed-set stage would maximize hexane extract yields.     

Amelioration of ionizing radiation induced lipid peroxidation in mouse liver by Moringa oleifera Lam. leaf extract

Protective effect of Moringa oleifera leaf extract (MoLE) against radiation-induced lipid peroxidation has been investigated. Swiss albino mice, selected from an inbred colony, were administered with MoLE (300 mg/kg body wt) for 15 days before exposing to a single dose of 5 Gy 60Co-gamma radiation. After treatments, animals were necropsied at different post irradiation intervals (days 1, 7 and 15) and hepatic lipid peroxidation and reduced glutathione (GSH) contents were estimated to observe the relative changes due to irradiation and its possible amelioration by MoLE. It was observed that, MoLE treatment restored GSH in liver and prevented radiation induced augmentation in hepatic lipid peroxidation. Phytochemical analysis showed that MoLE possess various phytochemicals such as ascorbic acid, phenolics (catechin, epicatechin, ferulic acid, ellagic acid, myricetin) etc., which may play the key role in prevention of hepatic lipid peroxidation by scavenging radiation induced free radicals.     

Fractionation and characterization of polysaccharides from abaca fibre

Abaca fibre polysaccharides were fractionated into water soluble, pectic, 1% NaOH soluble, hemicellulosic and cellulose fractions by extraction with hot water, dilute hydrochloric acid (pH 1.6), aqueous 1% NaOH and 17.5% NaOH, respectively. Cellulose (60.4–63.6%) and hemicelluloses (20.8%) were the major polysaccharides in abaca fibres. The hot water soluble polysaccharides contained noticeable amounts of pectic substances and a large proportion of neutral polysaccharides. The pectic polysaccharide preparation was enriched in both galacturonic acid and neutral sugars, including xylose, glucose, galactose, arabinose, and rhamnose. Extraction of the fibre with aqueous 1% NaOH produced the hemicellulose–lignin complex, which was enriched in xylose and, to a lesser extent, glucose-, arabinose- and galactose-containing polysaccharides, together with 7.6% associated lignin. Further extraction of the delignified fibre residue with aqueous 17.5%. NaOH removed the hemicellulose fractions, which were strongly enriched in xylose-containing polysaccharides. Besides ferulic and p-coumaric acids, six other phenolic monomers were also detected in the mixtures of alkaline nitrobenzene oxidation of associated lignin in all the polysaccharide fractions. The content of bound lignin in water soluble, pectic, and 1% NaOH soluble polysaccharides (Fractions 1, 2, and 3), isolated directly from the lignified fibres, was 12 times that of the hemicellulosic preparations (Fractions 4 and 5) isolated from the delignified fibre residues.     

Mitogenic Activity of Cytoplasmic Membranes Isolated from L-Forms of Staphylococcus aureus

Cytoplasmic membranes of L-forms of Staphylococcus aureus exerted a strong mitogenic effect on splenocytes of athymic nude mice as well as normal mice, while a cytoplasmic fraction of the same bacteria did not show definite mitogenicity. The mitogenic principle(s) of the membrane fraction was resistant to treatment with trypsin and was heat stable (at 100 C for 10 min). The active principle(s) in the insoluble residue of the membrane fraction digested with trypsin was not extracted with cold acetone, but could be solubilized by extraction with a cold chloroform-methanol mixture (2:1, v/v). The mitogenic principle(s) in the extract was fractionated by silicic acid column chromatography. Among five fractions separated by chromatography, fractions eluted with chloroform-methanol mixtures (1:1 and 1:20, v/v) were found to be strongly mitogenic. The cytoplasmic membranes of the L-forms also exerted a definite mitogenic effect on guinea pig splenocytes, but not on the thymocytes.     

Induction of chlorosis, ROs generation and cell death by a toxin isolated from Pyricularia oryzae

The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.     

NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences

An NAD(P)H oxidase activity stimulated by phenolic compounds has been investigated in purified plasma membranes (pm) and in an intracellular membrane (icm) fraction depleted in plasma membranes, both obtained from a microsomal fraction from cauliflower inflorescences ( Brassica oleracea L.). The phenolic compounds salicylhydroxamic acid (SHAM), ferulic acid, coniferyl alcohol, n -propyl gallate, naringenin, kaempferol and caffeic acid all strongly stimulated the activity. Peroxidase (EC 1.11.1.7), or a peroxidase-like enzyme, was responsible for the NAD(P)H oxidase activity, which proceeded through a free-radical chain reaction and was inhibited by catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and KCN. Most of the total activity was soluble; however, the membrane-bound activity was highly enriched in the pm compared to the icm. The catalase activity was 6 times higher in the icm-fraction than in the pm-fraction, but this was not the reason for the much lower phenol-stimulated NADH oxidase activity in the icm. Peroxidase activity measured with o -dianisidine and H₂O₂ had about the same specific activities in the pm-and icm-fractions.
Neither the phenol-stimulated NADH oxidase nor the peroxidase activity could be washed away from the pm even by 0.7 M NaCl, indicating that these activities are truly membrane-bound. SHAM as well as the other phenolic compounds capable of stimulating the NADH oxidase reaction were potent inhibitors of blue light-induced cytochrome b -reduction in the pm fraction.     

Antibacterial activity directed isolation of compounds from Onosma hispidum

The chemical investigation of the ethanolic extract of the root bark of Onosma hispidum following antibacterial activity directed isolation led to the isolation of 4-hydroxy-3-methoxy cinnamic acid (ferulic acid) and 4-hydroxy-3-methoxy benzoic acid (vanillic acid) which have been reported for the first time in this species. In addition to these compounds, the crude ethanolic extract and methanol fraction exhibited substantial bioactivity against species of corynebacteria, enterococci, staphylococci and streptococci. Ferulic acid was found more bioactive (being relatively more hydrophobic) compared to vanillic acid.     

Intake of phytochemicals among Japanese, calculated by the new FFF database

Effects of phytochemicals on human health are suggested from various animal experiments, but human studies remain insufficient. We have constructed a database of various phytochemicals (polyphenols, carotenoids, and sulphur compounds) (http://www.life-science.jp/fff/) and estimated the amount of intake among Japanese population. The subjects were volunteers (16 males and 63 females, averagely aged 71 and 61, respectively) in Iwate city. Average BMI was 23 in both sexes. Intake of 36 phytochemicals was calculated from one-day dietary records of all intake, by multiplying concentrations of each phytochemical in foods. Phytochemicals with average intake of at least 10 micromole per day were catechin, isoflavones, isothiocyanate, ferulic acid, quercetin, cinnamic acid and chlorogenic acid. Chief component analysis yielded 12 factors (80%).     

An ecofriendly approach to process rice bran for high quality rice bran oil using supercritical carbon dioxide for nutraceutical applications

An integrated approach to extraction and refining of RBO using supercritical carbon dioxide (SC-CO2) in order to preserve the nutritionally important phytochemicals is reported here. Process variables such as pressure, temperature, time, solvent flow rate and packing material on extraction yield and quality of RBO were investigated using a pilot model SC-CO2 extraction system. Three isobaric (350, 425 and 500 bar), three isothermal temperatures (50, 60 and 70 degrees C), three extraction times (0.5, 1 and 1.5h), at 40/min CO2 flow rate and three packing materials (pebbles, glass beads and structured SS rings) were employed. The RBO yield with SC-CO2 extraction increased with temperature and time under isobaric conditions. At the 60 degrees C isotherm, an increase in the RBO yield was obtained with an increase in the pressure and time. The RBO yield increased significantly with structured SS rings used as packing material. The RBO extracted with SC-CO2 had negligible phosphatides, wax and prooxidant metals (Fe and Cu) and was far superior in color quality when compared with RBO extracted with hexane. At the optimum condition of extraction at 500 bar, 60 degrees C for 1.5h, with structured SS rings used as packing material, the yield of RBO was comparable with that of hexane extraction (22.5%). The phytochemical contents of the RBO under the optimum conditions were in the range of tocols, 1500-1800 ppm; sterols, 15,350-19,120 ppm and oryzanol 5800-11,110 ppm.     

Bioproduction and Purification of Rubratoxin

Methods were developed for bioproduction and extraction of rubratoxin B from liquid cultures of Penicillium rubrum P-13 (NRRL A-11785). A maximum of 874.7 mg of toxin per liter of medium was attained in 21 days using stationary cultures of Mosseray's simplified Raulin solution enriched with 2.5% malt extract. Malt extract was required for rubratoxin production. Rubratoxin was not produced in either shake flasks or in fermentors with restricted aeration. Crystalline toxin was obtained by liquid-liquid extraction of concentrated culture medium with ethyl ether. Adhering colored impurities were removed by column chromatography and by recrystallization from acetone.     

Microbial transformation of ferulic acid to vanillic acid by <Emphasis Type="Italic">Streptomyces sannanensis</Emphasis> MTCC 6637

Streptomyces sannanensis MTCC 6637 was examined for its potentiality to transform ferulic acid into its corresponding hydroxybenzoate-derivatives. Cultures of S. sannanensis when grown on minimal medium containing ferulic acid as sole carbon source, vanillic acid accumulation was observed in the medium as the major biotransformed product along with transient formation of vanillin. A maximum amount of 400 mg/l vanillic acid accumulation was observed, when cultures were grown on 5 mM ferulic acid at 28°C. This accumulation of vanillic acid was found to be stable in the culture media for a long period of time, thus facilitating its recovery. Purification of vanillic acid was achieved by gel filtration chromatography using Sephadex™ LH-20 matrix. Catabolic route of ferulic acid biotransformation by S. sannanensis has also been demonstrated. The metabolic inhibitor experiment [by supplementation of 3,4 methylenedioxy-cinnamic acid (MDCA), a metabolic inhibitor of phenylpropanoid enzyme 4-hydroxycinnamoyl-CoA ligase (4-CL) along with ferulic acid] suggested that biotransformation of ferulic acid into vanillic acid mainly proceeds via CoA-dependent route. In vitro conversions of ferulic acid to vanillin, vanillic acid and vanillin to vanillic acid were also demonstrated with cell extract of S. sannanensis. Further degradation of vanillic acid to other intermediates such as, protocatechuic acid and guaiacol was not observed, which was also confirmed in vitro with cell extract.     

Modification of OGUR-ROSEN method for estimation of RNA in fresh and wounded sweet potato root tissues and change in RNA contents after the cutting

The suitability of the OGUR-ROSEN method for RNA extractionfrom sweet potato root tissue was investigated. The PCA extractfrom the freshly sliced tissue was colorless and showed a typicalspectrum of RNA. However, as the slices were incubated for 1to 3 days (wounded tissues), extract from them was brownishand revealed no typical spectrum of RNA. When wounded tissuewas treated with acetone containing isoascorbic acid and theresultant acetone powder was treated with PCA, extract indicatedthe spectrum very close to that of RNA. Interfering substancesstill remaining were removed satisfactorily with charcoal whichadsorbed RNA selectively. Thus, RNA could be assayed as theOD₂₆₀-difference between PCA extract from the acetone powderand a fraction of the extract non-adsorbable on charcoal. Timecourse analysis of RNA contents during incubation of the tissueslices disclosed a remarkable increase in the total RNA in theinitial stage of incubation. ¹This paper constitutes part 81 of Phytopathological Chemistryof Sweet Potato with Black Rot and Injury (Received September 5, 1969; )