Structural characterization and surface activity of hydrophobically functionalized extracted pectins

The technological properties of pectins are generally influenced by their chemical modification. Thus, amidated pectins are important derivatives with good emulsifying properties at low concentrations. The present article focuses on the comparative study of physicochemical properties of three modified pectin derivatives. Various amphiphilic derivatives in which pectin is associated with hydrophobic amines chains were prepared. The reaction was carried out in heterogeneous medium in methanol at 20 °C for 7 days and with 0.5 pectin/alkylamine mass ratio. The degrees of amidation (DA) of the derivatives were calculated based on the results of FTIR spectroscopy. The surface-active properties of the modified pectins were determined by surface tension (air/water) and interfacial tension (oil/water) measurements. The aminolysis of pectins appears to be an interesting way to produce pectin derivatives with new properties able to stabilize oil-in-water emulsions.     

Effect of pectin and amidated pectin on cholesterol homeostasis and cecal metabolism in rats fed a high-cholesterol diet

Two experiments were performed to compare the effect of pectin and its hydrophobic derivatives on homeostasis of cholesterol and cecal metabolism in male young rats. Control rats were fed a diet supplemented with palm fat and cholesterol (50 and 10 g/kg, respectively). Rats of other groups were fed the same diet containing citrus pectin or octadecylpectinamide (60 g/kg). Diets were fed for 4 weeks. In experiment I, pectinamide of lower degree of amidation (30 %) increased serum HDL cholesterol from 1.20 to 1.43 micromol/ml (p>0.05) at the expense of other cholesterol fractions. In experiment II, pectinamide of a higher degree of amidation (53 %) significantly decreased total serum cholesterol from 2.08 to 1.67 micromol/ml. Amidated pectins at both levels of substitution significantly decreased hepatic concentrations of cholesterol and fat. In both experiments the relative weight of cecum in the pectinamide group was significantly lower than in pectin group. The highest cecal concentrations of short-chain fatty acids (SCFA) were found in rats fed a diet with pectin (133.2 and 129.3 micromol/g in experiment I and II, respectively). In other groups, cecal SCFA was significantly (pectinamide groups) or non-significantly (controls) lower. In wet feces, SCFA concentrations were higher and butyrate molar proportions lower than in corresponding cecal contents. Pectinamide of a lower or higher degree of substitution significantly increased fecal content of cholesterol from 18.5 and 17.3 micromol/g in controls to 31.8 and 28.0 micromol/g, respectively. Corresponding concentrations of coprostanol were decreased. Effects of pectin on cholesterol homeostasis were absent or marginal. Histological examination revealed that hepatic tissue of control and pectin-fed rats was infiltrated with lipids. The Sudan black-positive material was absent in the liver of rats fed pectinamides. No pathological changes of liver tissue were apparent. In summary, hydrophobic amidated pectins significantly altered cholesterol homeostasis in rats and might be considered as a clinically effective hypocholesterolemic agent. Low cecal SCFA concentrations in rats fed pectinamides suggest that amidation of pectin had decreased its fermentability.     

Degree of blockiness of amide groups as indicator for difference in physical behavior of amidated pectins

Thickening and gelling properties of commercial amidated pectins depend on the degree of amidation and methyl-esterification, but also the distribution of these groups is of great importance. Methods have been developed during the last few years to determine the distribution of methyl esters over the pectic backbone. We applied the strategies developed for the analysis of high methyl-esterified pectins for studying the distribution of amide groups in amidated pectins. Low methyl-esterified amidated (LMA) pectins were digested before and after removal of methyl esters by an endo-polygalacturonase to determine the degree of blockiness of the substituents. The nature of the substituents (amide groups compared to methyl esters) did not modify the behavior of the enzyme. Oligomers released were separated by using high-performance anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD) at pH 5. Fractions collected after on-line desalting were identified by using MALDI-TOF mass spectrometry. Oligomers were found to elute from the column as a function of their total charge. For the same overall charge and size, oligomers with methyl esters eluted before oligomers with amide groups. Both amide groups and methyl esters of the LMA pectins studied were found to be semirandomly distributed over the pectic backbone, but this may vary according to the amidation process used.     

Complexation of amidated pectin with poly(itaconic acid) as a polycarboxylic polymer model compound

Complexes based on amidated pectin (AP) and poly(itaconic acid) (PIA) were prepared by casting films from solutions of AP and PIA in different ratios with the pectin amount ranging from 10% to 90% by mass. The complexes were investigated by elemental analysis, Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and thermogravimetry (TG). In all investigated ratios of AP/PIA glassy transparent films with a uniform structure were obtained. The results of elemental analysis confirmed the composition of the complexes, and FTIR spectroscopy has shown carboxylic and amide peak shifting, indicating complex formation between AP and PIA. Comparison of thermograms of AP/PIA films with different ratios of AP indicated that the increase of the amount of AP increases the thermal stability of the films by retarding the onset of the main degradation processes.     

Esters and amides of 2,3-dimethoxy-8,9-methylenedioxy-benzo[i]phenanthridine-12-carboxylic acid: potent cytotoxic and topoisomerase I-targeting agents

The exceptional topoisomerase I-targeting activity and antitumor activity of 5-(2-N,N-dimethylamino)ethyl-8,9-dimethoxy-2,3-methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one (ARC-111, topovale) prompted studies on similarly substituted benzo[i]phenanthridine-12-carboxylic ester and amide derivatives. Among the benzo[i]phenanthridine-12-carboxylic esters evaluated, the 2-(N,N-dimethylamino)ethyl, 2-(N,N-dimethylamino)-1-methylethyl, and 2-(N,N-dimethylamino)-1,1-dimethylethyl esters possessed similar cytotoxicity, ranging from 30 to 55 nM in RPMI8402 and KB3-1 cells. Several of the carboxamide derivatives possess potent topoisomerase I-targeting activity and cytotoxicity. The 2-(N,N-dimethylamino)ethyl, 2-(N,N-diethylamino)ethyl, and 2-(pyrrolidin-1-yl)ethyl amides were among the more cytotoxic benzo[i]phenanthridine-12-carboxylic derivatives, with IC₅₀ values ranging from 0.4 to 5.0 nM in RPMI8402 and KB3-1 cells.     

Rheology of Mixed Pectin Solutions

The effects of sucrose (S) and pectin (P) concentrations and the ratio between two distinct pectins (R) on the rheological behavior of diluted pectin systems were evaluated simultaneously using the surface response methodology. The systems were composed of a mixture of two high methoxy pectins with different degree of methyl esterification values (HM1/HM2) and of a mixture of a high-methoxy with an amidated low-methoxy pectin (HM1/LMA). For the HM1/HM2 systems, the multivariate analysis showed that the sucrose and pectin concentrations exerted statistically significant (p < 0.05) linear effects on the consistency index k and viscosity, the influence of pectin being about five times higher than that of sucrose. The pectin concentration and the ratio between the different pectins were shown to be significant with respect to the rheological parameters of the HM1/LMA systems. Evaluating the influence of the ratio between the different pectins, a synergistic effect on the structure reinforcement was observed when mixing HM1 and LMA in similar proportions, indicating the importance of the presence of hydrophobic interactions between methyl ester groups in addition to the stronger hydrogen bonding in junction zone stabilization. In general, the conditions in which hydrogen bonds were favored in relation to hydrophobic interactions led to systems with higher pseudoplasticity.     

Synthesis of 2-(2-adamantyl)piperidines and structure anti-influenza virus A activity relationship study using a combination of NMR spectroscopy and molecular modeling

The 2-(2-adamantyl)piperidines 13 and 15a-c were synthesized and evaluated for anti-influenza virus A and B activity. The parent N-H compound 13 was 3–4 times more active than amantadine and rimantadine against H₂N₂ influenza A. N-alkylation of 13 resulted in derivatives 15a-c that were devoid of biological activity. This dramatic reduction in biological activity may be attributed to the different conformational properties between N-H and N-alkyl piperidines, as deduced from the combination of computational chemistry and NMR spectroscopy.     

Chemoselective enzymatic preparation of N-hydroxyalkylacrylamides, monomers for hydrophilic polymer matrices

A lipase-catalyzed procedure is described for the preparation of N-hydroxyalkylacrylamides useful among a number of electrophoretical applications such as capillary and gel electrophoresis. The N-hydroxyalkylacrylamides were prepared through an aminolysis reaction of alkanolamines on ethyl acrylate. The reaction was catalyzed by Candida antarctica lipase. The addition of radical inhibitors improved chemoselectivity and amides were obtained in high yield and purity at room temperature.     

The quantitative analysis of uronic acid polymers by infrared spectroscopy.

I.r. absorption bands associated with the functional groups of carboxylic acid derivatives are useful for the analysis of alginates and pectins. The ester, amide, and uronate contents of pectins and the uronate content of alginates were determined, respectively, from the ester-carbonyl stretching band (1740 cm- minus 1), the amide I band (1650 cm- minus 1), and the carboxylate antisymmetric stretching band (1607 cm- minus 1) obtained from the spectra of solutions in D2O-phosphate buffer. The results are accurate to within plus or minus 2-4%, are self consistent, and agree well with the few reliable results that are available. The method should be applicable for the determination of carboxylic acid derivatives in other polysaccharides.     

Synthesis of UV-curable chitosan derivatives and palladium (II) adsorption behavior on their UV-exposed films

Novel chitosan derivatives with UV-curable functional groups, such as 3-methoxy-4-(2-hydroxy-3-methacryloyloxypropoxy)benzyl, 3,4-bis(2-hydroxy-3-methacryloyloxypropoxy)benzyl, 3-methoxy-4-methacryloyloxybenzyl, and 3,5-dimethacryloyloxybenzyl groups, were prepared. Introduction of photosensitive functional groups to chitosan was accomplished by reductive N-alkylation via Schiff’s bases using corresponding photosensitive aldehydes. Compared to starting chitosan, UV-curable chitosan derivatives showed better solubility in several organic solvents, such as DMSO and 70% methacrylic acid. The solubility of these compounds increased with an increase in the degree of substitution of the N-alkyl side chains. After UV irradiation for 20 s under a high-pressure mercury lamp at a distance of 15 cm from the samples, acidic methanol solutions of these derivatives were transformed to gels in the presence of photo-initiator, and their dried films adsorbed palladium (II) at pH 1.1 and pH 5.3. The UV-curable chitosan derivatives were successfully used as coating materials for electroless plating on non-conductive substances.     

Kinetic characterization of carboxypeptidase-Y-catalyzed peptide semisynthesis Prediction of yields

Summary Carboxypeptidase-Y-catalyzed peptide semisynthesis has been characterized at pH 7.5, 25°C from initial rate steady state kinetic and progress reaction studies of hydrolysis and aminolysis of-N-benzoyl-L-tyrosine 4-nitro-anilide using the natural L-amino acids and their amides as nucleophiles. The reaction mechanism previously shown to account for carboxypeptidase-Y-catalyzed aminolysis reactions (Christensen et al., 1992) was found also to account for all of the reactions studied here. It involves in addition to the classical serine proteinase mechanism: i) complex formation between the free enzyme and the nucleophile, an interaction characterized by the competitive inhibition constant,K _i, and ii) reaction of the nucleophile with the acylated enzyme forming a complex of enzyme and aminolysis product, characterized by the aminolysis kinetic parameter,K _N.A competitive inhibitory effect showing binding to the free enzyme is seen mainly with large hydrophobic amino acids and their amides i.e. the same residues as those preferred on either side of the scissile bond in carboxypeptidase-Y substrates. The stoichiometry of the inhibition is 1 : 1 and the actual binding position most likely is that of the leaving group of substrates,S ₁.Aminolysis effects are obtained with a wide range of amino acids and amino acid amides, exceptions are Pro and, probably due to their low solubility, Tyr, Trp, Asp and Glu. TheK _N-values show relatively little dependence on the chemical nature of the side groups, but a marked difference between the amino acid and its amide. The amides interact more strongly. The kinetic parameter,k _c/K_m, of the hydrolysis of the aminolysis products is another important factor in peptide semisynthesis. Thek _c/K_m-values obtained of the amidated aminolysis products are much less than those of the products formed with free amino acids. All in all this leads to rather efficient aminolysis with the L-amino acid amides and poor aminolysis with the L-amino acids.Abbreviations BzTyrNHPhNO₂ -N-benzoyl-L-tyrosinyl 4-nitro-aniline - Xaa L-amino acids - Xaaa L-amino acid amides - Z-Phe Carbobenzoxy-L-phenylalanine - Z-Met Carbobenzoxy-L-methionine - BzTyr -N-benzoyl-L-tyrosine - AlaVal L-alanyl-L-valine - ValAla L-valyl-L-alanine     

Synthesis of sulfated pectins and their anticoagulant activity

The following pectins were sulfated: bergenan BC (the pectin of Bergenia crassifolia L), lemnan LM (the pectin of Lemna minor L), and galacturonan as a backbone of pectins. Pyridine monomethyl sulfate, pyridine sulfotrioxide, and chlorosulfonic acid were used as reagents for sulfation. Chlorosulfonic acid proved to be the optimal reagent for sulfation of galacturonan and other pectins. Galacturonan and pectin derivatives with different degrees of sulfation were synthesized and their anticoagulant activities were shown to depend on the quantity of sulfate groups in the pectin macromolecules.     

Evaluation of aminolysis of anilinothiazolinones to phenylthiocarbamoyl amino acid methyl amides as an alternative conversion method in protein sequencing.

The aminolysis of products of sequential degradation of proteins and peptides by methylamine is an alternative method of conversion of the unstable 5-alkyl-2-anilino-4-thiazolinones into the stable methyl amides of N alpha-phenylthiocarbamoyl amino acids. The volatility of methylamine permits use in the gas phase during both manual and automatic sequential degradation. Two procedures were studied: (mode A) aminolysis by methylamine in the sequencer reaction chamber after liberation of the thiazolinones by trifluoroacetic acid and (mode B) aminolysis by methylamine vapors passed through a 1-chlorobutane solution of thiazolinones in the conversion flask of the sequencer. The sequencing program was modified for both procedures by making use of the standard sequencer functions. The yields of aminolysis in the conversion flask (mode B) are comparable to those obtained by standard conversion in 25% trifluoracetic acid and the procedure does not affect the repetitive yield. Aminolysis on the glass filter (mode A) requires a major modification of the degradation process, yet gives higher yields of the degraded amino acid derivatives. A disadvantage of both procedures, especially of mode A, is the presence of N-methyl-N'-phenylthiourea in the methyl amide samples. We have not been able to achieve the expected improvement of the yields of degraded hydroxy amino acids. Therefore the replacement of acid conversion of anilinothiazolinones to phenylthiohydantiones by aminolysis for routine degradation cannot be recommended. High yields of methyl amides make aminolysis a promising candidate for the incorporation of fluorescent or other labels in the products of sequencing degradation.     

Action pattern of Valencia orange PME de-esterification of high methoxyl pectin and characterization of modified pectins

Valencia pectinmethylesterase (PME) fractions, B-PME, containing 36 and 13 kDa protein bands and U-PME, containing a 36 and 27 kDa protein bands, were used to de-esterify original pectin (O-Pec) from 73% degree of esterification (%DE) to 63% (B-Pec) and 61% DE (U-Pec), respectively. Most O-Pec eluted from ion exchange chromatography at low salt concentration and a smaller component eluted at higher ionic strength. B-Pec and U-Pec eluted as one broad peak at higher ionic strength. PME modification did not change molecular weight: O-pectin (134,000 g/mol), U-Pec (133,850 g/mol), and B-Pec (132,250 g/mol). The NMR signal of GG and GGG increased after modification, whereas the signal of EE and EEE decreased. The negative zeta-potential increased with pH for all pectins. U-PME and B-PME created differently modified pectins that vary in degree and length of multiple attacks and fraction of the pectin population that was modified.     

Benzotriazole stabilized lithium intermediates as a route to the elaboration of N-substituents in amides

Lithiation of N-(benzotriazol-1-ylmethyl)benzamide or N-(benzotriazol-1-ylmethyl)-2,2-dimethylbutyramide, readily prepared from the corresponding amides, formaldehyde and benzotriazole, followed by quenching with various electrophiles, such as alkyl halides, ketones or ester, gives the corresponding N-substituted derivatives. Subsequent displacement of the benzotriazole group with Grignard reagents, thiols or alcohols provides access to a wide variety of N-substituted amides in good yields. Treatment of the N-(benzotriazol-1-ylalkyl)benzamides with n- BuLi afforded the 1,1-dibenzamidoalkanes.     

Comparative effect of amidated pectin and psyllium on cholesterol homeostasis in rats

The effects of amidated pectin and psyllium on serum, hepatic and faecal cholesterol concentration were compared in female rats fed diets supplemented with palm fat and cholesterol at 50 and 10 g/kg, respectively. Control rats were fed a diet supplemented with cellulose at 60 g/kg. In treated rats, cellulose was replaced with either amidated pectin or psyllium. Amidated pectin and psyllium intake significantly decreased serum cholesterol from 3.41 μmol/ml (control) to 1.68 and 2.04 μmol/ml, respectively, and hepatic cholesterol from 31.9 μmol/g (control) to 7.2 and 9.0 μmol/g, respectively. Histology with lipid-staining Sudan Black B revealed that liver tissue from control rats was infiltrated with lipids, but staining was absent in livers of treated rats. No hepatic pathophysiology was apparent in treated rats. Amidated pectin and psyllium intake significantly increased faecal fat content. Faecal cholesterol content was significantly increased in rats that were fed amidated pectin, and non-significantly increased in rats that were fed psyllium. Body weight and food intake did not differ among treatment groups. In conclusion, amidated pectin, a novel sequestrant of sterols, demonstrated a similar effect on rat serum and hepatic cholesterol concentration to psyllium, which is a well-established hypocholesterolaemic agent.     

Alpha-1-C-alkyl-1-deoxynojirimycin derivatives as potent and selective inhibitors of intestinal isomaltase: remarkable effect of the alkyl chain length on glycosidase inhibitory profile

A series of - and β-1-C-alkyl-1-deoxynojirimycin derivatives was prepared and evaluated as glycosidase inhibitors. Biological assays showed a marked dependence of the selectivity and potency of the inhibitors upon the position of the alkyl chain (-1-C-, β-1-C- or N-alkyl derivatives). In addition, the efficiency of -1-C-alkyl-1-deoxynojirimycin derivatives as intestinal isomaltase inhibitors increases with the length of the alkyl chain. The strongest inhibition was found for -1-C -nonyl-1-deoxynojirimycin with an IC₅₀ = 3.5 nM (25× more potent inhibitor than the shorter chain homologue carrying a C₈ chain). These results demonstrate that subtle changes in the aglycon fragment may result in remarkable enzyme specificity.     

Functional characterization of the gels prepared with pectin methylesterase (PME)-treated pectins

High- and low-methoxyl pectins were treated with pectin methylesterase (PME) and the functional properties of the resulting pectin gels were characterized. The degree of esterification of high- and low-methoxyl pectins decreased from 74.5% to 6.3% and 40.0% to 6.5%, respectively while not changing their molecular weight. Also, the addition of glucono-delta-lactone (GDL) dramatically affected the gel strength and the pH reduction by the GDL led to the increased syneresis of the pectin gels, which was also observed in the PME-treated samples. When flavor compounds were incorporated into the pectin gels, the flavor release from the gels increased with decreasing the degree of esterification due to increased hydrophilic properties.     

Composition and properties of pectin polysaccharides of St. John’s wort Hypericum Perforatum L.

Three fractions of acidic water-soluble polysaccharides (concentration of glucuronic acid 10?C65%) were obtained from the above-ground part of St. Johns wort Hypericum perforatum L. by serial extraction with water and 0.7% aqueous solution of ammonium oxalate. Enzymatic hydrolysis of these polysaccharides using endo-polygalacturonase indicates that their carbohydrate chains contain the units of galacturone formed by 1,4-??-linked residues of non-substituted D-galacturonic acid. The extracted polysaccharides have been purified by means of gel filtration. It has been shown that water-soluble polysaccharides obtained by extraction with water manly contain the residues of galactose, mannose, glucose, and arabinose (the concentration of glucuronic acid being 10?C27%) while the polysaccharide fraction extracted using 0.7% aqueous solution of ammonium oxalate is presented by pectin polysaccharides. Only the residues of galacturonic acid (55?C72%) have been identified among glucuronic acids in its composition using chromatography/mass spectrometry of trimethylsilyl derivatives. In addition, this fraction contains the residues of the neutral monosaccharides which are typical for pectins: arabinoses, galactoses, rhamnoses, and glucose; there are also minor concentrations of residues of xylose and mannose. IR spectra of pectin polysaccharides of St. John??s wort have absorption bands in the ranges 1740, 1640?C1620, 1236?C1200, and 1200?C1000 cm^?1 which are typical for pectins. It has been demonstrated that aqueous solutions of pectin polysaccharides of St. John??s wort (2 mg/mL) have pronounced antioxidant activity (44% of the activity of trolox taken for 100%).     

The potential of pectin as a stabilizer for liposomal drug delivery systems

The aim of the present study was to investigate the potential of different types of pectin as stabilizers for liposomal drug delivery systems. Positively charged liposomes were coated with commercially available and purified low-methoxylated (LM), high-methoxylated (HM) and amidated (AM) pectins. The samples were stored for up to 12 weeks at 4°C, at room temperature and at 35°C. The change in liposomal size and size distribution, zeta potential, pH, leakage of encapsulated carboxyfluorescein (CF), and lipid degradation were studied. All the types of pectin were found to protect the liposomes against aggregation during storage. The pectin coat did not affect the permeability of the liposome membrane. HM and LM pectin seemed to be the most promising types of pectin due to minimal changes in the zeta potentials during storage for these samples and no detectable lipid degradation. It is concluded that pectin may be used for stabilizing liposomal drug delivery systems.