In vivo studies of the biosynthesis of vernolic acid in the seed of Vernonia galamensis

In vivo radiotracer experiments using [1-¹⁴C]acetate as the precursor were conducted to investigate the biosynthesis of vernolic acid (12, 13-epoxy-cis-9-octadecenoic acid) in the seeds of Vernonia galamensis. The acetate precursor radioactively labeled vernolate in phosphatidylcholine (PC), diacylglycerol, and triacylglycerol. Time-course kinetics of the incorporation of the radioactive tracer indicated that vernolate is synthesized while the acyl moiety is esterified to PC. Pulsechase experiments provided additional supporting evidence that vernolate is synthesized while esterified to PC. These results are consistent with the hypothesis that linoleoyl PC is the precursor of vernoleoyl-PC. Subsequently, vernolate is quickly moved from the PC pool to the triacylglycerol pool, where it accumulates.     

Vernonia anthelmintica (L.) Willd. trivernolin, 1,3-divernolin and vernolic (Epoxyoleic) acid from the seed oil

The epoxy fatty acid components isolated from the seed oil ofV. anthelmintica, Indian ironweed, where the seed had been allowed to undergo lipolysis after grinding, were trivernolin, 1,3-divernolin, and vernolic acid. By inactivation of the hydrolytic enzyme system present in the seed, oil containing more than 50% trivernolin may be obtained. This species has potentialities as a replacement crop for those now in surplus; its seed contains 20 to 26% of an oil rich in epoxyoleic (vernolic) acid combined as glycerides amounting to 70 to 75%. Presented at the A.O.C.S. meeting in St. Louis, 1961. Eastern Utilization Research and Development Division, Agricultural Research Service, U.S.D.A.     

Vernonia anthelmintica willd. Seed oil and salts of vernolic acid as stabilizers for plasticized poly (Vinyl chloride)

Vernonia anthelmintica seed oil, trivernolin, the main constituent of the oil, barium and cadmium salts of vernolic acid (epoxyoleic acid), and various combinations of the salts have been evaluated as heat and light stabilizers of plasticized poly (vinyl chloride). Evaluation was made at the 1 and 3% levels of the vernonia oil components. Transparent sheets whose color ranged from light amber to colorless were obtained after milling and molding. All compounds studied greatly improved both heat and light stability of the sheets; the 3% stabilizer level gave the better results. In general the stabilizing ability of the compounds studied increased as follows: vernonia oil<trivernolin<barium vernolate< cadmium vernolate and <barium-cadmium vernolate mixtures. Comparisons with 3 commercial stabilizers show that the vernonia oil and derivatives thereof are equal to or better than the comparative commercial controls. Presented at the 35th Fall Meeting of the American Oil Chemists' Society, Chicago, Ill. Eastern Utilization Research and Development Division, Agricultural Research Service, U.S.D.A.     

Syntheses of 12-aminododecanoic and 11-aminoundecanoic acids from vernolic acid

12-Aminododecanoic acid and 11-aminoundecanoic acid, monomer precursors for nylon-12 and nylon-11, respectively, have been synthesized from vernolic (cis-12,13-epoxy-cis-9-octadecenoic) acid via a reaction sequence that includes the formation of 12-oxododecanoic acid oxime. Saponification of vernonia oil, followed by a low-temperature recrystallization at −20°C, gave 51% vernolic acid (97% purity, m.p. 23–25°C). Hydrogenation afforded cis-12,13-epoxystearic acid (m.p. 52–54°C, lit. m.p. 52–54°C), which upon oxidation with periodic acid in tertiary butyl alcohol gave 12-oxododecanoic acid with an isolated yield of 71.0%. Reaction of the oxoacid with hydroxylamine hydrochloride gave 12-oxododecanoic acid oxime, which was catalytically reduced to give 12-aminododecanoic acid with a yield greater than 85% and a melting point of 184–186°C (lit. m.p. 185–187°C). 11-Aminoundecanoic acid was prepared from the 12-oxododecanoic acid oxime via a three-step reaction sequence that involved a Beckmann rearrangement, Hofmann degradation, and hydrolysis. Thus, the aldoxime acid was hydrolyzed in the presence of nickel acetate tetrahydrate to give 11-carbamoylundecanoic acid (48% yield, m.p. 129–131°C, lit. m.p. 129–130°C). The amide was then treated with a solution of sodium methoxide and bromine at 70–80°C to give 11-(methoxycarbonylamino)undecanoic acid at 75% yield (m.p. 84–86°C; elemental analysis, calculated for C₁₃H₂₅NO₄: C, 60.19; H, 9.73; N, 5.40; O, 24.68%; found C, 60.02; H, 9.81; N, 5.26; O, 24.91%), which upon alkaline hydrolysis and subsequent neutralization gave 11-aminoundecanoic acid at 34% yield (m.p. 189–192°C, lit. m.p. 190°C). Mass spectrometric and ¹³C nuclear magnetic resonance data of the previously unreported 11-(methoxycarbonylamino)undecanoic acid is provided.     

Generation and Characterization of a Soybean Line with a Vernonia galamensis Diacylglycerol Acyltransferase-1 Gene and a myo-Inositol 1-Phosphate Synthase Knockout Mutation

Soybean (Glycine max) meal is an important protein source. Soybean meal with lower phytate and oligosaccharides improves meal quality. A single recessive mutation in soybean myo-inositol 1-phosphate synthase (Gm-lpa-TW75-1) confers a seed phenotype with low phytate and increased inorganic phosphate. The mutant was crossed with high oil lines expressing a diacylglycerol acyltransferase1 (DGAT) gene from Vernonia galamensis (VgD). Gm-lpa-TW75-1 X VgD, designated GV, has 21%, and 22% oil and 41% and 43% protein from field and greenhouse seed production, respectively. No significant differences were found in mineral concentrations except for Fe which was 229 μg/g dry mass for GV followed by 174.3 for VgD and 162 for Gm-lpa-TW75-1. Phosphate (Pi) is higher in Gm-lpa-TW75-1 as expected at 5 mg/g, followed by GV at 1.6 mg/g whereas Jack, VgD, and Taiwan75 have about 0.3 mg/g. The Gm-lpa-TW75-1 line has the lowest phytate concentration at 1.4 mg/g followed by GV with 1.8 mg/g compared to Taiwan75, VgD, and Jack with 2.5 mg/g. This work describes a high oil and protein soybean line, GV, with increased Pi and lower phytate which will increase the nutritional value for human and animal feed.     

Fumaric acid production from hydrolysates of starch-based substrates

Fumaric acid production by Rhizopus arrhizus from commercial hydrolysates of corn starch (i.e. glucose molasses) was studied at different initial concentrations of glucose (S) and C:N ratios (R) by performing a 3² factorial experiment. By using the response surface methodology and statistical analysis, fumaric acid (Y_F) and mycelial biomass (Y_x) yields, as referred to the initial concentration of glucose and fumaric acid productivity (P_F), were fitted to the only significant first-order effects of S and R with mean percentage errors ranging from 11 to 15%. The resulting empiric models were used to determine the optimal values of S (100–130 g dm^?3) and R (150–210 g-atom C per g-atom N) associated with Y_F and P_F varying in the ranges 40–49% and 7–8.5 g dm^?3 day^?1, respectively. After establishing the validity of these data at the 95% confidence level, an optimal operating condition (S = 120 g dm^?3 and R = 150) was further tested using other substrates (i.e. glucose and acid or enzymatic hydrolysates of cassava, corn and potato flours). Statistically significant improvements in the fumaric acid yield and productivity were determined with respect to the predicted values. Since the highest values of Y_F and P_F were obtained from the acid hydrolysates of the starch-based materials and such values were also found to be insensitive to the substrate used (at a probability level of 0.05), the above operating condition might be further employed to minimise fumaric acid production costs as a function of the feedstock used.     

Synthesis and reactivity of antioxidants based on vernolic acid and 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionic acid

The reactivity of sterically hindered phenols toward free radicals of the hydrocarbon cumene was studied. New compounds, i. e. methyl cis-13(12)-{3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionyloxy}-12(13)-hydroxyoleate and cis-13(12)-{3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionyloxy}-12(13)-hydroxyoleic acid were synthesized starting from vernolic acid and its methyl ester, respectively, and 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionic acid. Both new compounds possess antioxidant activities similar to those of commercially available antioxidants.     

Determination of vernolic acid content in the oil ofEuphorbia lagascae by gas and supercritical fluid chromatography

Determination of the content of vernolic acid (12,13-epoxy-9c-octadecenoic) in the oil ofEuphorbia lagascae has been performed by gas chromatography of the fatty acid methyl ester derivatives of the triacylglycerols in the oil and by supercritical fluid chromatography (SFC) of the raw oil and the fatty acid derivatives of the oil. The content of vernolic acid was found to be 55 wt%. The three methods were compared, and SFC analysis of the fatty acid derivatives was found to be the most accurate method.     

Tailored and deep porosification of LTCC substrates with phosphoric acid

Low temperature co-fired ceramics (LTCC) as an advanced technology for robust assembly of electronic components, has attracted significant attention in a wide application range such as in wireless communication or automotive radar systems. However, accurate designs of micromachined devices operated at high frequencies require substrates with regions of tailored permittivities. Introduction of controlled porosity into the substrate via wet-chemical etching procedure, is a promising approach for permittivity reduction which can be applied to commercially available LTCC without necessitating to alter their composition or sintering process. In the present study, by selective dissolution of celsian phase a very deep porosification (highest reported so far) could be realized while preserving the surface quality. Also, by a careful selection of the etching parameters, the depth of porosification and hence the permittivity reduction can be delicately tailored. Laser ablation inductively coupled plasma mass spectrometry was used for the investigation of chemical compositions of substrates.     

Glyceride studies: Part IX: Intraglyceride distribution of vernolic acid and of five conjugated octadecatrienoic acids in seed glycerides

Vernolic (epoxyoleic) acid, present in six seed oils over the range 19–72%, appears to be preferentially attached to the secondary glycerol hydroxyl group. The distribution of five isomeric conjugated octadecatrienoic acids (8, 10, 12 and 9, 11, 13) in 18 seed oils has been examined by lipase-catalyzed deacylation. The results are not entirely consistent and more species must be examined before a rational distribution pattern becomes apparent.     

The use of lipase (acetone powder) from Vernonia galamensis in the fatty acid analysis of seed oils

Examined in this study is a potential application of Vernonia galamensis lipase (acetone powder) in enzyme-catalyzed hydrolysis of seed oils, most especially of those containing reactive functionalities which are easily affected under drastic hydrolytic and methylating/transesterification conditions during fatty acid analysis. Eight seed oils from V. galamensis, Ximenia kaffra, castor, corn, soyabean, palm kernel, sunflower and olive were hydrolyzed by lipase (acetone powder) followed by methylation using diazomethane in ethyl ether. Results obtained showed that the lipolytic hydrolysis of triglycerides by V. galamensis lipase (acetone powder) was probably nonspecific and did not result in isomerization and as such hydrolyzed the triglycerides of the seed oil in a fashion that the resulting fatty acids were unaltered during hydrolysis. The fatty acids obtained were representative of the parent seed oils. Values reported for the various seed oils were similar to those of previous studies.     

Molecular Docking and Molecular Dynamics Simulation Studies of Triterpenes from Vernonia patula with the Cannabinoid Type 1 Receptor

A molecular docking approach was employed to evaluate the binding affinity of six triterpenes, namely epifriedelanol, friedelin, α-amyrin, α-amyrin acetate, β-amyrin acetate, and bauerenyl acetate, towards the cannabinoid type 1 receptor (CB1). Molecular docking studies showed that friedelin, α-amyrin, and epifriedelanol had the strongest binding affinity towards CB1. Molecular dynamics simulation studies revealed that friedelin and α-amyrin engaged in stable non-bonding interactions by binding to a pocket close to the active site on the surface of the CB1 target protein. The studied triterpenes showed a good capacity to penetrate the blood–brain barrier. These results help to provide some evidence to justify, at least in part, the previously reported antinociceptive and sedative properties of Vernonia patula.     

Antinociceptive and Anti-Inflammatory Effects of Ethanol Extract from Vernonia polyanthes Leaves in Rodents

The ethanol extract from Vernonia polyanthes leaves (EEVP) was investigated for antinociceptive and anti-inflammatory effects at the doses (p.o.) of 100, 200 and 400 mg/kg in animal models. The extract reduced the number of abdominal contortions by 16.75% and 31.44% at a dose of 200 and 400 mg/kg, respectively. The results obtained showed that EEVP exerted a significant antinociceptive effect in the two phases of formalin. The EEVP increased the reaction time on a hot plate at the doses of 100, 200 and 400 mg/kg after 90 min of treatment. The paw edema was reduced by EEVP at the doses of 100, 200 and 400 mg/kg after 4 h of application of carrageenan. Doses of 200 and 400 mg/kg, administered 4 h before the carrageenan injection, significantly reduced the exudate volume (29.25 and 45.74%, respectively) and leukocyte migration (18.19 and 27.95%, respectively). These results suggest that V. polyanthes can be an active source of substances with antinociceptive and anti-inflammatory activities.     

Nanoparticle removal from substrates with pulsed-laser induced plasma and shock waves

Removing particles with nanometer scale diameters from substrates is a challenging task with numerous critical applications. A novel removal method for nanoparticles is developed and tested. The technique, which is dry and non-contact, takes advantages of shock wavefronts initiated by plasma formation under a focused laser beam pulse and its interaction with the substrate. Experimental results indicate that silica particles down to 500 nm on silicon wafers can be removed without substrate damage. In the reported experiments, a Q-switched Nd : YAG pulsed laser with a 5-ns pulse width and 360-mJ pulse energy at 1064 nm wavelength is employed as a plasma generation source. It is reported that the traditional dry laser cleaning method based on the rapid thermal expansion under direct laser irradiation often results in surface damage in the nanometer scale due to light diffraction around nanoparticles and/or stress localization in the thermal skin. This occurs when the characteristic dimensions of the particles are comparable to the wavelength of incident beam. In the laser plasma method, various mechanisms are responsible for the removal effect. The strong shock wave in air generates complex pressure wavefields resulting in both drag/lift on the particle and acceleration of the substrate. However, shock waves are not transmitted to the solid substrate due to a large difference between the relevant wave phase speeds in the two media. The effects of the number of shots and the distance between the surface and the plasma boundary on the removal efficiency are reported.     

Stereospecificity of monoacylglycerol and diacylglycerol acyltransferases from rat intestine as determined by chiral phase high-performance liquid chromatography

Using chiral phase high-performance liquid chromatography of diacylglycerols, we have redetermined the ratios of 1,2-/2,3-diacyl-sn-glycerols resulting from acylation of 2-monoacylglycerols by membrane bound and solubilized triacylglycerol systhetase of rat intestinal mucosa. With 2-oleoyl[-³H]glycerol as the acyl acceptor and oleoyl-CoA as the acyl donor, 97–98% of the diacylglycerol product was 1,2(2,3)-dioleoyl-sn-glycerol, 90% of which was thesn-1,2-and 10% thesn-2,3-enantiomer. The remaining diacylglycerol (less than 3%) was thesn-1,3-isomer. The overall yield of acylation products was 70%, of which 60% were diacylglycerols and 40% triacylglycerols. With 2-oleylglycerol ether as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, 90% of the diradylglycerol was 1-oleoyl-2-oleyl-sn-glycerol and 10% was the 2-oleyl-3-oleoyl-sn-glycerol. The diradylglycerols made up 96% and the triradylglycerols 4% of the radioactive product. With 1-palmitoyl-sn-glycerol as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, the predominant reaction product was 1-palmitoyl-3-oleoyl-sn-glycerol. The 3-palmitoyl-sn-glycerol was not a suitable acyl acceptor. Both 1,2- and 2,3-diacyl-sn-glycerols were substrates for diacylglycerol acyltransferase as neither isomer was favored when 1,2-dioleoyl-rac-[2-³H]glycerol was used as the acyl acceptor. There was a marked decrease in the acylation of the 1(3)-oleoyl-2-oleyl-sn-glycerol to the 1,3-dioleoyl-2-oleyl-sn-glycerol. It is concluded that neither monoacylglycerol nor diacylglycerol acyltransferase exhibit absolute stereospecificity for acylglycerols as fatty acid acceptors.     

New approaches to clarify antinociceptive and anti-inflammatory effects of the ethanol extract from Vernonia condensata leaves

The present study was aimed at evaluating the antinociceptive and anti-inflammatory effects of the ethanol extract from Vernonia condensata leaves in animal models, in order to afford a better understanding of these properties. The extract reduced the number of abdominal contortions at doses of 100 (51.00 ± 3.00), 200 (42.00 ± 2.98) and 400 mg/kg (39.00 ± 4.00). In formalin tests, a significant reduction in the licking time (p < 0.01) was observed in the first phase by 25.14 (200 mg/kg = 51.50 ± 4.44) and 31.15% (400 mg/kg = 48.00 ± 4.37). The doses of 100 (43.37 ± 5.15), 200 (34.62 ± 4.16) and 400 mg/kg (28.37 ± 3.98) inhibited (p < 0.001) the second phase. After 60 and 90 min of treatment, a dose of 400 mg/kg (10.13 ± 0.39 and 11.14 ± 1.33, respectively) increased the latency time. Doses of 200 and 400 mg/kg potentiated the sleeping time induced by diazepam, pentobarbital and meprobamate. The extracts (100, 200 and 400 mg/kg) showed anti-inflammatory effects by a decrease in paw edema. The extracts also reduced the exudate volume at the doses of 200 and 400 mg/kg. The leukocyte migration had significant effect (p < 0.001) at doses of 100, 200 and 400 mg/kg. The completion of additional experiments in the investigation of the antinociceptive and anti-inflammatory activities of V. condensata allowed a better understanding of the central and peripheral mechanisms involved.     

Vernonia oil–based acrylate and methacrylate polymers and interpenetrating polymer networks with epoxy resins

Acrylate and methacrylate monomers were obtained by reacting vernonia oil, a naturally epoxidized oil, with acrylic or methacrylic acid. The highest conversion (85–98%) of epoxy groups was obtained when the reaction was performed with an excess of the carboxylic acid at 100–120°C. The acrylate and methacrylate monomers of vernonia oil were characterized by IR and NMR spectroscopy. These monomers were then cured by sunlight in the presence of benzophenone to produce transparent films. In addition, interpenetrating polymer neworks (IPNs) were prepared in a two‐step technique from the sunlight‐cured methacrylate of vernonia oil, as the elastomeric component, in combination with a cured epoxy resin (a bisphenol A–type resin). Dynamic mechanical analysis showed good compatibility between the networks of the two cured polymers. An IPN with a 1 : 1 composition of the two polymer components exhibited the properties of a reinforced elastomer. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 91: 3835–3843, 2004     

<Emphasis Type="Italic">Geranium sanguineum</Emphasis> (Geraniaceae) seed oil: A new source of petroselinic and vernolic acid

The occurrent of petroselinic acid (18∶1Δ6cis) in seed oils was believed to be limited to the Umbelliferae or Apiaceae, and a few other members of the Umbelliflorae. A major occurrence of petroselinic acid outside the Umbelliflorae must therefore be regarded as highly unusual and surprising. The seed oil of Geranium sanguineum, a member of the family Geraniaceae, has now been found to contain petroselinic and vernolic acids as major FA in its seed oil TAG. These unusual FA have not been reported previously as constituents of Geraniaceae seed oils. The structure and composition of the seed oil FA from G. sanguineum were determined by combined use of chromatographic (TLC, capillary GLC) and spectroscopic (IR, GC-MS) techniques. The double-bond position in petroselinic acid was located unambiguously by the characteristic mass fragmentation of its dimethyldisulfide (DMDS) adduct. The epoxy FA was identified as vernolic acid by co-chromatography and by the mass fragments formed during GC-MS of the products of the epoxy ring-opening reaction with BF₃ in methanol.     

Biochemical Properties of a New Cold-Active Mono- and Diacylglycerol Lipase from Marine Member Janibacter sp. Strain HTCC2649

Mono- and di-acylglycerol lipase has been applied to industrial usage in oil modification for its special substrate selectivity. Until now, the reported mono- and di-acylglycerol lipases from microorganism are limited, and there is no report on the mono- and di-acylglycerol lipase from bacteria. A predicted lipase (named MAJ1) from marine Janibacter sp. strain HTCC2649 was purified and biochemical characterized. MAJ1 was clustered in the family I.7 of esterase/lipase. The optimum activity of the purified MAJ1 occurred at pH 7.0 and 30 °C. The enzyme retained 50% of the optimum activity at 5 °C, indicating that MAJ1 is a cold-active lipase. The enzyme activity was stable in the presence of various metal ions, and inhibited in EDTA. MAJ1 was resistant to detergents. MAJ1 preferentially hydrolyzed mono- and di-acylglycerols, but did not show activity to triacylglycerols of camellia oil substrates. Further, MAJ1 is low homologous to that of the reported fungal diacylglycerol lipases, including Malassezia globosa lipase 1 (SMG1), Penicillium camembertii lipase U-150 (PCL), and Aspergillus oryzae lipase (AOL). Thus, we identified a novel cold-active bacterial lipase with a sn-1/3 preference towards mono- and di-acylglycerides for the first time. Moreover, it has the potential, in oil modification, for special substrate selectivity.     

Radiotagged soils. Removal from several substrates and adsorption site character

Substrates investigated were glass, quartz, porcelain, steel, stainless steel, aluminum, polyethylene, methylmethacrylate, Nylon, and Teflon. Radiotagged (C-14) soils used were algal protein, stearic acid, and tristearin. Soil removal curves showed that Nylon, Teflon, methacrylate, and stainless steel had few soil-adsorption sites and that the adsorption was of an ion-exchange type. Algal protein and stearic acid soils appeared to adsorb through an ion-exchange type mechanism, with most substrates, while tristearin showed a van der Waals' type of adsorption with glass, quartz, steel, and aluminum. Conclusions as to adsorption type were based upon the shape of the soil-removal curves. Adsorption studies showed that both anionic and nonionic surfactants were adsorbed by glass, procelain, steel, and aluminum surfaces, apparently by an ion-exchange type of adsorption. The character of the surfactant adsorbed affected the degree of removal of subsequently applied soil; the more hydrophilie surfactants permitted easier soil-removal. Tripolyphosphate, orthophosphate, and ethylenediamine tetraacetate anions and tetraethylammonium cations were adsorbed by these same substrates and influenced the ease of removal of subsequently applied soil. Pretreatment of substrate by alkali generally increased the ion or surfactant adsorption more than acid-pretreated surfaces, and rather marked similarities between the adsorption sites of glass and quartz were apparent from acid pretreatment. Stearic acid soiled substrates were cleaned by a preferential displacement mechanism; the soil was rolled up into globules. Tristearin-soiled surfaces were cleaned by a similar mechanism.