Reversed-phase HPLC Profiles Research Articles

Synthesis and characteristic of 1-amino-2-propanol-based ionic liquids and effective extraction of antioxidants from Mugwort

Green extraction of natural products from plants is of high importance to the pharmaceuticals and fine chemicals industry. Novel bio-renewable ionic liquids, a class of safe and powerful medium, has aroused broad attention from the field of extraction and separation. In the present study, the 1-amino-2-propanol-based ionic liquids (PA-ILs) were used to selectively extract the antioxidants from Mugwort (Artemisia argyi Lévl. et Vant.). As revealed from the results, among ten PA-ILs carrying different counter anions, the PA/mandelic acid, PA/EDTA and PA/lactic acid exhibited the best extraction efficiency to polyphenols (120.34 mg/g), flavones (128.87 mg/g) and polysaccharides (43.62 mg/g), respectively. Furthermore, the time-dependent DPPH radical scavenging shown that flavones are the major antioxidants and PA-ILs extractants were higher than those of conventional solvents. According to the reversed-phase HPLC profiles, more polar substances (e.g. glycosides) existed in ILs extract compared with alcohols solvent. The Fermi resonance in the FT-IR spectrum indicated the complex H-bonding framework in ILs, which may act as a crucial factor to assess extraction performance. Moreover, the in vitro studies reported that the cytotoxicity of PA-ILs for HaCaT and Caco-2 cell lines was less than that of pure PA. It was, therefore, demonstrated that PA-ILs are a secure medium that can help alternate organic solutions.

Geographical variability of the venoms of four populations of Bothrops asper from Panama: Toxicological analysis and neutralization by a polyvalent antivenom

Bothrops asper is the medically most important venomous snake in Central America. In Panama, the country having the highest incidence of snakebites in Latin America, B. asper is widely distributed throughout the country and is responsible for the vast majority of snakebites. This study was performed to analyze whether there are variations in the toxicological profile and in some biochemical parameters between the venoms of B. asper from four different regions in Panama. The venoms showed a similar profile of lethal, hemorrhagic, in vitro coagulant, defibrinogenating, edema-forming, myotoxic and indirect hemolytic activities, with subtle quantitative variations between samples of some regions. The venoms also had similar SDS-PAGE patterns and reverse phase HPLC profiles. A polyvalent antivenom manufactured in Costa Rica, and regularly used in Panama, was effective in the neutralization of lethal activity of the venoms of the four populations, with Mean Effective Doses (ED50) ranging from 5.98 to 9.72 mg venom/mL antivenom. In agreement, a widespread pattern of cross-reactivity between this antivenom and the four venoms was observed by immunoblotting. Overall, results highlight the lack of marked differences between the venoms of the various populations of B. asper in Panama, and that the antivenom from Costa Rica is effective in neutralizing lethality.

살 오징어(Todarodes pacificus) 간췌장으로부터 aminopeptidase 활성 획분의 쓴맛 개선 효과

Aminopeptidase-active fractions from crude extract of the hepatopancreas of a common squid (Todarodes pacificus) were obtained using acetone (AC; 30-40%) and ammonium sulfate precipitation (AS; 60-70% saturation), anion exchange (AE-II; 0.2 M NaCl) and gel filtration chromatography (GF-I; 30-50 kDa), respectively. The debittering capacity of GF-I fraction based on the aminopeptidase activity (89.2 U/mg), recovery (56.6%) and sensory evaluation (1.0) was better than that of other fractions. Release of amino acids increased as incubation time was increased, and the bitterness of the enzyme reaction mixtures decreased. Incubation with the GF-I fraction for 24 h resulted in the hydrolysis of several peptides, as revealed by reverse-phase HPLC profiles. Peaks 3, 5 and 6 showed the decreased area (%), whereas peaks 1, 2 and 4 showed the increased area. The GF-I fractions were found to be suitable for reducing bitterness in protein hydrolysates by catalyzing the hydrolysis of bitter peptides.

Use of dairy and non-dairy Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus strains as adjuncts in cheddar cheese

Lactobacilli have been used as adjunct cultures in the manufacture of different cheeses with the objective of accelerating ripening and/or improving cheese quality, but no studies have been conducted with strains from non-dairy origins. A miniature cheddar-type cheese model was used to screen ten dairy and non-dairy Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus strains for their performances as adjuncts in cheese manufacture. All strains were able to grow and survive in the cheese environment and produced only minor, although statistically significant, changes in gross cheese composition. Adjuncts affected secondary proteolysis causing differences in the levels of free amino groups, total free amino acids and reversed-phase HPLC (RP-HPLC) profiles of pH 4.6-soluble extract. Three strains were selected on the basis of differences in proteolysis pattern and used in a pilot-plant production of cheddar cheese, which was ripened for 180 days. The results confirmed that use of L. plantarum adjuncts significantly affected secondary proteolysis as measured by free amino acid production with minor impact on gross composition and primary starter performance, but the impact on RP-HPLC profiles of pH 4.6-soluble extracts was not statistically significant. The use of a strain originally isolated from olive brine fermentation, L. plantarum P1.5, resulted in significantly improved preference scores over the control.

Cross‐Neutralisation of the Neurotoxic Effects of Egyptian Cobra Venom with Commercial Tiger Snake Antivenom

Cross-neutralisation has been demonstrated for haemorrhagic venoms including Echis spp. and Cerastes spp. and for Australia elapid procoagulant toxins. A previous study showed that commercial tiger snake antivenom (TSAV) was able to neutralise the systemic effects of the Egyptian cobra, Naja haje, in vivo but it is unclear if this was true cross-neutralisation. The aim of the current study was to determine whether TSAV can neutralise the in vitro neurotoxic effects of N.haje venom. Both Notechis scutatus (10μg/ml) and N.haje (10μg/ml) venoms caused inhibition of indirect (supramaximal V, 0.1Hz, 0.2msec.) twitches of the chick biventer cervicis nerve-muscle preparation with t(90) values (i.e. the time to produce 90% inhibition of the original twitch height) of 26±1min. (n=4) and 36±4min.; (n=4). This effect at 10μg/ml was significantly attenuated by the prior addition of TSAV (5U/ml). A comparison of the reverse-phase HPLC profiles of both venoms showed some similarities with peak elution times, and SDS-PAGE analysis elucidated comparable bands across both venoms. Further analysis using Western immunoblotting indicated TSAV was able to detect N.haje venom, and enzyme immunoassay showed that in-house biotinylated polyclonal monovalent N.scutatus antibodies were able to detect N.haje venom. These findings demonstrate cross-neutralisation between different and geographically separated snakes supporting potential immunological similarities in snake toxin groups for a large range of snakes. This provides more evidence that antivenoms could be developed against specific toxin groups to cover a large range of snakes.

Effect of sonication on thermolysin hydrolysis of ovotransferrin

The effect of sonication on proteolytic hydrolysis of ovotransferrin and bioactivities of the hydrolysates were investigated, and the large peptide fragments left in the hydrolysate were characterised. The results showed that sonication could increase the reactive sulphydryl groups in 5% ovotransferrin solution by 50%, although there were no improvements in the overall degree of hydrolysis. Furthermore, SDS–PAGE and reverse-phase HPLC profiles did not show difference of the treated samples. Angiotensin-converting enzyme (ACE) inhibitory activity, but not antioxidant activity, of the thermolysin hydrolysate was improved in a dose dependent manner at prolonged sonication time. Matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) analysis revealed that thermolysin hydrolysate was composed of 926 peptides, of 99% having masses lower than 10 kDa and of 1% larger than 10 kDa derived from both N- and C-lobes of ovotransferrin.

Linking lenticel discolouration of mango (Mangifera indica L.) fruit to reversed-phase HPLC profiles of phenolic compounds

SummaryDiscolouration of mango lenticels intensifies progressively from a slight red halo during the initial development of the condition, to a blackened area surrounding each lenticel. Initial aqueous methanol extracts of discoloured tissue were further fractionated into phenolic groups and analysed by means of reversed-phase high performance liquid chromatography. Discernable shifts in phenolic profiles correlated with the colour changes in affected lenticels. After staining with Vanillin-HCl, fluorescence microscopy of fixed material revealed the locations of, and changes in the different forms of phenolics associated with various degrees of discolouration. The results confirmed that the compounds responsible for the discolouration of lenticels were simpler flavonones rather than more complex anthocyanidins. The chromatographic results correlated with previous observations using transmission electron microscopy, which indicated that the accumulation of cell wall-bound phenolics did not result from vacuolar collapse or membrane disintegration.

Synthesis and structural study of variously oxidized diastereomeric 5′‐dimethoxytrityl‐thymidine‐3′‐O‐[O‐(2‐cyanoethyl)‐N,N‐diisopropyl]‐phosphoramidite derivatives. Comparison of the effects of the PO, PS, and PSe functions on the NMR spectral and chromatographic properties

R(P)- and S(P)-diastereomers of 5'-dimethoxytrityl-thymidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropyl]-phosphoramidite (T-CED) were separated by silica gel chromatography. Oxidation of both isomers with H(2)O(2), elemental sulfur and selenium, respectively, resulted in the corresponding oxidized analogues in nearly quantitative yields. All reactions were found to proceed with retention of P-configuration. This was confirmed by thorough NMR analysis which, in addition, aimed to study the spectral properties of the diastereomers with special respect to differences in the heteroatom effect of the O, S and Se atoms, double-bonded to the phosphorus, on the vicinal carbon-phosphorus couplings. It was found that the changes in the DeltaJ (=(3)J(P,C4') - (3)J(P,C2')) values were basically induced by the electronegativity of the heteroatoms, rather than differences in the rotational preferences about the C3'-O3' bond. The impact of the benzene solvent on the above couplings is also discussed. The effect of these heteroatoms on the chromatographic (normal and reverse phase HPLC) behavior of the compounds was also investigated and the reverse phase HPLC profiles showed an unambiguous correlation between the electronegativity of the heteroatoms and the chromatographic mobility of the analogues.

The linoleic acid derivative DCP-LA selectively activates PKC-ɛ, possibly binding to the phosphatidylserine binding site

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.

Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

During injury or trauma, blood coagulation is initiated by the interaction of factor VIIa (FVIIa) in the blood with freshly exposed tissue factor (TF) to form the TF.FVIIa complex. However, unwanted clot formation can lead to death and debilitation due to vascular occlusion, and hence, anticoagulants are important for the treatment of thromboembolic disorders. Here, we report the isolation and characterization of two synergistically acting anticoagulant proteins, hemextins A and B, from the venom of Hemachatus haemachatus (African Ringhals cobra). N-terminal sequences and CD spectra of the native proteins indicate that these proteins belong to the three-finger toxin family. Hemextin A (but not hemextin B) exhibits mild anticoagulant activity. However, hemextin B forms a complex (hemextin AB complex) with hemextin A and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Complex formation was supported by size-exclusion chromatography. Using a "dissection approach," we determined that hemextin A and the hemextin AB complex prolong clotting by inhibiting TF.FVIIa activity. The site of anticoagulant effects was supported by their inhibitory effect on the reconstituted TF.FVIIa complex. Furthermore, we demonstrated their specificity of inhibition by studying their effects on 12 serine proteases; the hemextin AB complex potently inhibited the amidolytic activity of FVIIa in the presence and absence of soluble TF. Kinetic studies showed that the hemextin AB complex is a noncompetitive inhibitor of soluble TF.FVIIa amidolytic activity, with a Ki of 50 nm. Isothermal titration calorimetric studies showed that the hemextin AB complex binds directly to FVIIa with a binding constant of 1.62 x 10(5) m(-1). The hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require a scaffold to mediate its inhibitory activity. Molecular interactions of the hemextin AB complex with FVIIa/TF.FVIIa will provide a new paradigm in the search for anticoagulants that inhibit the initiation of blood coagulation.

Effect of artisanal liquid rennet from kids and lambs abomasa on the characteristics of Feta cheese

Feta cheeses were manufactured with commercial calf rennet and artisanal liquid rennet from kids and lambs abomasa with an aim to investigate the influence of rennet type on the characteristics of this cheese variety. The two rennets had similar chymosin to pepsin ratios and they were used in quantities with the same total milk coagulating activity. The use of traditional rennet had no significant effect on the evolution of physicochemical composition (pH, total solids, ash, NaCl, fat, total protein) or on the evolution of proteolysis (water-soluble nitrogen expressed as percentage of total nitrogen, free amino acids expressed as mmol Leu, residual α s-casein and reversed phase HPLC profiles) during the ripening. The textural characteristics of mature 60d-old cheeses were not significantly different. The microbiological quality of traditional rennet significantly affected the enterococci and coliform counts of 3d-old cheeses but this influence was eliminated in the mature 60d-old cheeses. The significantly higher flavour scores of mature Feta made with traditional artisanal liquid rennet were in accordance with the significantly higher C4:0 and C10:0 FFA contents resulting from the lipase activity of traditional rennet.

Functionality of Bacillus proteinase hydrolysates of sodium caseinate

Sodium caseinate (NaCN) was digested with Protamex, a Bacillus proteinase, at 40°C and pH 7.0 to degree of hydrolysis (DH) values of 2.7%, 5.3% and 13.3%. The solubility, emulsifying, foaming and viscosity properties of the hydrolysates were investigated between pH 2.0 and 10.0. Foam expansion of >1300% was observed for the 5.3% DH hydrolysate at pH 4.0, compared to 670% for unheated NaCN. Significantly improved foam expansion properties (P<0.005) were observed over the entire pH range examined for the 13.3% DH hydrolysate compared to unheated or heat-treated NaCN. Hydrolysis resulted in significantly improved solubility (P<0.005) around the isoelectric point and significant improvements in the emulsifying activity and stability (P<0.005) at alkaline pH compared to unheated NaCN. Hydrolysis with Protamex increased the apparent viscosity of NaCN at the isoelectric point. Reversed-phase HPLC profiles showed that high DH samples contained high levels of hydrophilic peptides.

CDNA cloning and antibacterial activities of cecropin D-like peptides from Agrius convolvuli.

We have characterized full-length cDNAs encoding two isoforms of agriusin, cecropin D-like antibacterial peptide, present in the hemolymph of the immunized Agrius convolvuli larvae. The cloned cDNAs of agriusins 1 and 2 contain 331 and 329 bp, respectively. The nucleotide sequencing of cDNAs showed that they encode 62 amino acids, whose mature portion was deduced to consist of 38 amino acid residues with over 94% sequence identity. In the sequence homology search, mature agriusin 1 showed over 86 and 71% amino acid sequence homology with bactericidin 4 from Manduca sexta and cecropin D from Hyalophora cecropia, respectively. Since it was demonstrated from the deduced amino acid sequences that the C-terminal residues of agriusins are followed by a Gly residue, two types of synthetic agriusin 1 (syn-agriusin 1 amide and acid) were prepared to verify if natural agriusin 1 is C-terminally amidated. From acid-urea PAGE and reversed phase HPLC profiles to compare two synthetic peptides, we could confirm that the C-terminal amino acid residue of natural agriusin 1, like several cecropins so far identified, is amidated. Finally, our antibacterial assay performed with two syn-agriusins 1 revealed that there is little difference between antibacterial activities of both peptides against Gram-positive and Gram-negative bacteria.

On shelf stability of freeze-dried poly(methylidene malonate 2.1.2) nanoparticles

Freeze-dried poly(methylidene malonate 2.1.2) (PMM 2.1.2)-made nanoparticles were evaluated for their 12-month stability under various storage conditions of temperature and illumination. At different period of time after their preparation, nanoparticles were resuspended and tested for their size, and the molecular weight and reversed-phase HPLC profiles of their components. The suspension pH, its turbidity at 400 nm as well as its in vitro cytotoxicity on CEM T cell line were also measured. Generally, nanoparticles maintained at 40°C underwent significant alterations revealed by the suspension pH decrease, the progressive modification of the HPLC chromatogram and the decreased cytotoxicity. Degradation of the polymer side chains and generation of carboxyl moieties could account for these observations. From the 1 year measurements here reported, lyophilized PMM 2.1.2 colloidal nanoparticles conserved at room temperature or below, either in darkness or in daylight, may be assumed to have a satisfactory shelf-life.

Hydroperoxide-mediated cytochrome P450-dependent 8-anilino-1-naphthalenesulfonic acid destruction, product formation and P450 modification.

The interaction between hydroperoxides, cytochrome P450 and 8-anilino-1-naphthalenesulfonic acid (ANS) has been investigated. The addition of ANS to the cytochrome P450 solution did not effect the P450 Soret absorption peak or the reduced CO difference spectrum, suggesting that ANS may not bind to P450 home directly. H2O2 or CuOOH alone did not effect ANS fluorescence and absorption spectra indicating that no detectable reaction occurs between hydroperoxide and ANS in the absence of P450. The reconstituted system of cytochrome P450, P450 reductase, lipid and NADPH did not mediate ANS metabolism. In the presence of P450, the addition of either H2O2 or CuOOH, however, leads to a decrease in ANS absorption around 258 nm and 350 nm indicating possible destruction of ANS. ANS destruction was confirmed with the disappearance of the ANS elution peak in the reverse phase HPLC profiles and with the changes in P450-bound ANS fluorescence intensity and the shift of lambda max of ANS. Moreover, a very sensitive method to detect trace fluorescent products of ANS by thin layer chromatography has been developed based on the fact that ANS fluorescence is enhanced more than 1000-fold by the organic solvent butanol. A UV-sensitive fluorescent product was detected on thin layer chromatography profiles of the reaction mixtures. P450 was also observed to be modified by a fluorescent derivative of ANS, when the fluorescence was enhanced by butanol. These results also show that an organic compound which can not be metabolized by the reconstituted system of cytochrome P450 and NADPH-P450 reductase is metabolized by the reconstituted system of P450 and hydroperoxide, suggesting the activities of these two systems may not be completely comparable.

Purification of natural killer-like Kurloff cells and arachidonic acid metabolism

Pulmonary and splenic Kurloff cells have been purified from estrogen-treated guinea pig. Enzymatic digestion of lung tissues and mechanical dispersion of cells yielded about 650 × 10 6 viable cells. After centrifugal elutraition and centrifugation on continuous Percoll gradient, a population of high-density (1,100 g/ml) pulmonary Kurloff cells were obtained with high viability (≈ 99%) and purity (≈ 99%). Splenic Kurloff cells have been isolated by disruption of spleen tissue and centrifugation on continous Percoll gradient. High-density splenic Kurloff cells (150 × 10 6 cells per spleen) were also obtained with high purity (≈ 99%) and viability (≈ 99%). Pulmonary and splenic Kurloff cells were incubated with various concentrations of arachidonic acid (10, 30 and 100 μM) in the absence or presence of 2 μM ionophore A23187. With 10 μM arachiodonic acid the relative production of cyclooxygenase products was the following: TxB 2 > PGE 2 ≈ PGI 2. For an arachidonic acid concentration superior to 10 μM, the profile of release was PGE 2 ⪢ TXB 2 > PGI 2 Arachidonic acid metabolism through the 5-lipoxygenase pathway was also studied by incubating pulmonary or splenic Kurloff cells with 10 μM arachidonic acid in the absence of 2 μM ionophore A23187, or in some experiments, with 2.5 μM leukotriene A 4. Reverse phase HPLC profiles clearly indicated that high-density Kurloff cells did not express 5-lipoxygenase activity. However, these cells showed the ability to convert exogenous leukotriene A 4 into leukotriene B 4 suggesting the presence of LT4 A hydrolase activity. These data have been confirmed by a sensitive RIA method. This study constitutes the first report on the purification of pulmonary Kurloff cells and on arachinodic acid metabilism by these cells. The possible implications of Kurloff cells in various biological events are discussed.

Affinity chromatography of catalytic autoantibody to vasoactive intestinal peptide.

An autoantibody in human IgG that hydrolyzes vasoactive intestinal peptide (VIP) was identified. The IgG did not hydrolyze VIP unless an unidentified inhibitor was removed by dialysis. The VIP antibody was fractionated by affinity chromatography on immobilized VIP by using IgG without VIP-hydrolytic activity as the starting material. The affinity-purified antibody catalyzed the hydrolysis of VIP (nominal kcat/Km: 1.1 X 10(6) M-1 min-1). The values of Km for the affinity-purified antibody preparation and unfractionated IgG (110 nM and 112 nM) suggested relatively tight antibody-VIP binding. A comparison of the reverse phase HPLC profiles of antibody-treated [Tyr10-125I]VIP with that of synthetic [125I]VIP(1-16) suggested that unfractionated IgG and the affinity-purified antibody cleaved the same peptide bond in VIP (Gln16-Met17).