Obtaining and study of monosaccharide derivatives of low-molecular-weight chitosan

The possibility of obtaining monosaccharide derivatives of low-molecular-weight chitosan with the use of the Maillard reaction was studied. Chitosan derivatives (molecular weight, 24 and 5 kDa) obtained with glucosamine, N-acetyl galactosamine, galactose, and mannose with a substitution degree of 4-14% and a yield of 60-80% were obtained. Some physicochemical and biological properties of these derivatives were studied. We showed that monosaccharide derivatives of low-molecular-weight chitosan exhibited antibacterial activity. Chitosan at a concentration of 0.01% caused 100% death of bacteria B. subtilis and E. coil. The strongest antibacterial effect was exhibited by 24-kDa derivatives: only 0.02-0.08% of cells survived. These derivatives were two orders of magnitude more effective than the 5-kDa chitosan modified with galactose.     

Galactosylated derivatives of low-molecular-weight chitosan: Obtaining and properties

Chitosan, a binary heteropolysaccharide consisting of 2-acetamide-2-deoxy-β-D-glucopyranose and 2-amino-2-deoxy-β-D-glucopyranose residues linked in different proportions via β-glycosidic bonds. The presence of a primary amino group in the chitosan structure allows for the synthesis of various derivatives. The procedure of obtaining activated N-hydroxysuccinimide esters with the use of lactobionic acid was applied to obtain galactosylated derivatives of low-molecular-weight chitosan with a substitution degree varying from 8 to 23%. The properties of these derivatives (viscosity, solubility, and biodegradability) were studied. These derivatives are well soluble at pH values greater than the acidity constant of amino groups of chitosan (6.5). Broadening the pH range towards increase and the presence of galactose residues allows these derivatives to be used in working with biological objects.     

Study of the Protective Activity of Chitosan Hydrolyzate Against Septoria Leaf Blotch of Wheat and Brown Spot of Tobacco

Chitosan hydrolyzate containing low-molecular-weight chitosan (≤24 kDa) and its oligomers (≤1.2 kDa) has been obtained via chemical depolimerization of high-molecular-weight chitosan by nitric acid. The fractions of the obtained chitosan hydrolyzate have been characterized by high performance gel permeation chromatography and proton magnetic resonance. The test performed on detached leaves of wheat has shown that the hydrolyzate completely inhibits the development of Stagonospora nodorum, a casual agent of the septoria leaf blotch, at a concentration of 200 μg/mL. A similar test with detached tobacco leaves has shown that the hydrolyzate at a concentration of 100 μg/mL also inhibits the development of Alternaria longipes, which causes brown spot of tobacco, by 75%.     

Regenerating Activity and Antibacterial Effect of Low-Molecular-Weight Chitosan

The antibacterial activity of two low-molecular-weight chitosans (LMWCs) was studied using two wound cultures: E. coli 1157 and St. albus. Both LMWCs exhibited antibacterial affect. The would-healing effect of methylcellulose gels comprising LMWCs (72 or 11 kDa) was studied. It was shown that wound-healing properties of gels depend on the molecular weight and concentration of LMWCs. The healing activity of LMWCs was assessed judging by the wound half-closure time. The gels exhibiting the greatest healing rate contained 2% 72-kDa LMWC or 0.1% 11-kDa LMWC. The composition of the oligomeric fraction of the 11-kDa LMWC was studied by HPLC. It was shown that the oligomeric moiety of the 11-kDa LMWC represents a mixture of oligomers with a large admixture of monomers (30.82%).     

In vitro antifungal efficacy of <Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9642 chitosan-AgNPs composite against post-harvest disease of citrus fruits

Green and blue mold postharvest diseases are the most vital negative components influencing the local market of citrus fruits. Citrus fruits were collected, and fungi were isolated. Among the fungal isolates identified, Penicillium digitatum and Penicillium italicum recorded the highest occurrence of 39.5 and 25.6%, respectively. In this work, we extracted chitosan from Aspergillum niger ATCC 9642. Fourier transform infrared spectroscopy was utilized to confirm the functional groups of the obtained compound, which exhibited the main characteristic bands of O–H stretching at 3302 cm^-1, and C–O–C band at 1125 cm^-1. A. niger ATCC 9642 chitosan had the degree of deacetylation of 88.5%, a molecular weight of 1.8 × 10⁵ Da, and viscosity of 7.3 centipoises; these values were comparable to those for standard shrimp chitosan. Ultraviolet-visible light spectra revealed the presence of A. niger ATCC 9642 chitosan-AgNPs composite. Using antifungal and spore germination assays, it was found that this composite exhibited effective antifungal action against P. digitatum and P. italicum compared with a chitosan standard. In a comet assay, the percentage of tail DNA was considered as a parameter that indicated DNA damage. The comet parameter increased significantly (P < 0.05) with A. niger ATCC 9642 chitosan–AgNPs composite, and the increase was dose-dependent. The increase in the DNA damage positively correlated with the inhibition performance of the A. niger ATCC 9642 chitosan–AgNPs composite.     

In vitro antifungal activity of metal complexes of a quaternized chitosan derivative with copper ions

Antifungal activity of synthetic metal complexes of quaternized N-(propyl) chitosan derivatives with Сu(II) against yeastlike (Saccharomyces cereviseae, Rodothorula rubra, and Candida albicans) and mycelial fungi (Fusarium oxysporum, Alternaria alternata, Cladosporium herbarum) was studied. In vitro application (at 250?500 μg/mL) of the metal complex of quaternized N-(propyl) derivative of low-molecular chitosan with 53% substitution and 1.3% copper ions proved efficient against F. оxysporum, one of ten most common fungal plant pathogens. Water-soluble quaternized N-(propyl) chitosan derivatives with 40?58% degree of substitution were synthesized using glycidyltrimethylammonium chloride under optimally adjusted conditions. Metal complexes of the chitosan derivative with 53% degree of substitution with Сu(II) ions were obtained by dialysis. The quantity of copper ions in the metal complexes was determined by atomic emission spectrometry. The structure of chitosan derivatives was confirmed by spectral analysis (IR, ¹H NMR).     

Antibacterial Activity of Alkylated and Acylated Derivatives of Low–Molecular Weight Chitosan

A number of alkylated (quaternized) and acylated derivatives of low–molecular weight chitosan were obtained. The structure and composition of the compounds were confirmed by the results of IR and PMR spectroscopy, as well as conductometric titration. The effect of the acyl substituent and the degree of substitution of N-(2-hydroxy-3-trimethylammonium) propyl fragment appended to amino groups of the C₂ atom of polymer chains on antibacterial activity against typical representatives of gram-positive and gramnegative microorganisms (Staphylococcus epidermidis and Escherichia coli) was studied. The highest activity was in the case of N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC) with the maximal substitution (98%). The minimal inhibitory concentration of the derivative was 0.48 μg/mL and 3.90 μg/mL for S. epidermis and E. coli, respectively.     

Preparation of <Emphasis Type="Italic">in situ</Emphasis> Injectable Chitosan/Gelatin Hydrogel Using an Acid-tolerant Tyrosinase

An in situ injectable chitosan/gelatin hydrogel was formed under slightly acidic conditions (pH 4.0 ~ 4.5) using an acid-tolerant tyrosinase, tyrosinase-CNK. A homogeneous chitosan/tyrosinase-CNK solution was prepared in one part of a dual-barrel syringe, and highly soluble gelatin in distilled water was prepared in the other part of the syringe without any additional crosslinking materials. Chitosan/gelatin hydrogel was formed in situ by simple injection of the solutions at room temperature followed by curing at 37°C. However, conventional mushroom tyrosinase did not catalyze this permanent gel formation. Tyrosinase- CNK-catalyzed glycol chitosan/gelatin hydrogel was similarly formed by this in situ injection approach. The hydrogels exhibited a high swelling ratio of 20-fold their own weight, interconnected micropores with an average diameter of approximately 260 μm and in vitro biodegradability suitable for tissue engineering and drug delivery applications. These results showed that tyrosinase-CNK-mediated chitosan/gelatin hydrogel formation has remarkable potential for the development of novel formulations for in situ injectable gel-forming systems.     

Inhibition of lipase activity by low-molecular-weight chitosan

Inhibition of enzymatic activity of lipase (EC 3.1.1.3) from the fungus Candida rugosa and wheat (Triticum aestivum L.) germ by low-molecular-weight chitosan with an average molecular weight of 5.7 kDa in reactions of p-nitrophenyl palmitate cleavage was studied. Preincubation of lipases with chitosan, prior to addition of the substrate to solution, showed that equilibrium during the lipase-inhibitor complex formation was reached within 30 min. The inhibition constants for C. rugosa lipase and wheat germ lipase were 1.4 and 0.9 mM, respectively. The contribution of electrostatic interactions to the complex formation between chitosan and lipases is insignificant.     

Inhibition of lipase activity by low-molecular-weight chitosan

Inhibition of enzymatic activity of lipase (EC 3.1.1.3) from the fungus Candida rugosa and wheat (Triticum aestivum L.) germ by low-molecular-weight chitosan with an average molecular weight of 5.7 kDa in reactions of p-nitrophenyl palmitate cleavage was studied. Preincubation of lipases with chitosan, prior to addition of the substrate to solution, showed that equilibrium during the lipase-inhibitor complex formation was reached within 30 min. The inhibition constants for C. rugosa lipase and wheat germ lipase were 1.4 and 0.9 mM, respectively. The contribution of electrostatic interactions to the complex formation between chitosan and lipases is insignificant.     

Synthesis and antibacterial activity of some new benzo[5,6]chromeno[2,3-<Emphasis Type="Italic">d</Emphasis>]pyrimidines

The synthesis and antibacterial activity of some new benzo[5,6]chromeno[2,3-d]pyrimidine derivatives are described. The title compounds were obtained by the reaction of 1H-benzo[f]chromenes with aliphatic and aromatic amines. The structures of all newly synthesized compounds were confirmed by IR, ¹HNMR, ¹³C NMR, and NOESY experiments. The compounds exhibited potent antibacterial activity against gram-positive and gram-negative bacterial species. 10-Methyl-12-(4-hydroxyphenyl)-10,12-dihydro-11H-benzo[5,6]chromeno[2,3-d] pyrimidin-11-imine displayed greater antibacterial activity against gramnegative bacterial species than did ciprofloxacinandamoxicillin.     

Trofosides A and B and other cytostatic steroid-derived compounds from the far east starfish <Emphasis Type="Italic">Trofodiscus über</Emphasis>

Three new polar steroids identified as trofoside A, 20R,24S)-24-O-(3-O-methyl-β-D-xylopyranosyl)-3β,6α,8,15β,24-pentahydroxy-5α-cholestane, its 22(23)-dehydro derivative (trofoside B), and 15-sulfooxy-(20R,24S)-5α-cholestane-3β,6β,8,15α,24-pentaol sodium salt, were isolated fromTrofodiscus über starfish extracts collected in the Sea of Ohotsk. Two known compounds, trofoside A aglycone, (20R,24S)-3β,6α,8,15β,24-pentahydroxy-5α-cholestane, and triseramide, (20R,24R,25S,22E)-24-methyl-3β6α,8,15β-tetrahydroxy-5α-cholest-22-en-27-oic acid (2-sulfoethyl)amide sodium salt, were also found. The structures of the isolated polyoxysteroids were established from their spectra. Minimal concentrations causing degradation of unfertilized egg-cells of the sea-urchin Strongylocentrotus intermedius(C _min) and terminating the cell division at the stage of the first division (C _min embr.), as well as the concentrations causing 50% immobilization of sperm cells (OC₅₀) and inhibiting their ability to fertilize egg-cells by 50% (IC₅₀) were determined for the isolated compounds. Of three compounds highly toxic in embryos and sea-urchin sperm cells, the polyol with a sulfo group in the steroid core was the most active; two glycosides with monosaccharide chains located at C3 and C24 atoms were less toxic. Note that all the compounds with the spermiotoxic activities differently affected the embryo development. The positions of monosaccharide residues in the core considerably influence the compound activity. For example, both mono-and double chained glycosides with the monosaccharide fragment at C3 and fragments at C3 and C4 atoms are active against sea-urchin sperm cells and embryos, whereas the C24 glycosylated trofoside A does not affect embryos and displays a poor spermiotoxicity.     

Antimicrobial Efficacy of Methylated Lac Dye,an Anthraquinone Derivative

A natural red dye which is produced by the tiny insects Kerria lacca while feeding on host trees is popularly known as lac dye. Lac dye is a mixture of at least five closely related pure compounds all being anthraquinone derivatives designated as laccaic acid A, B, C, D and E. Anthraquinones isolated from different natural sources and reported to have potent antimicrobial activity. The lac dye, which is also a mixture of anthraquinone derivatives, is expected to exhibit antifungal and antibacterial activity. Lac dye cannot be used as antibacterial and antifungal agent due to its low water solubility and high polarity. Therefore, it is modified into its methyl derivative to enhance its bio-efficacy. Methylated lac dye is characterized with the help of TLC, UV–Vis spectroscopy and FT-IR, NMR analysis. An in vitro spore germination assay was carried out to evaluate the antifungal efficacy of methylated lac dye against some phytopathogenic fungi which commonly caused a various foliar diseases in crop plants viz., Alternaria solani, Curvularia lunata, Erysiphe pisi, Helminthosporium oryzae and Verticillium sp. Among the tested fungi, Verticillum sp. showed highest sensitivity, which showed 100% inhibition at 750 and 1000 µg/ml as compared to control. However, E. pisi an obligate parasite also showed varied sensitivity but at 1000 µg/ml showed 100% spore germination as compared to control. Methylated lac dye also showed strong antibacterial properties against Ralstonia solanacearum at very low concentration (40 and 50 µg/ml). Hence, lac dye may serve as potent antifungal and antibacterial agent in plant disease management.     

Monosaccharide profiling of silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis> L.) nervous system during development and aging

Glycoconjugates have various functions in differentiation, development, aging and in all aspects of normal functioning of organisms. The reason for increased research on this topic is that glycoconjugates locate mostly on the cell surface and play crucial biological roles in the nervous system including brain development, synaptic plasticity, learning, and memory. Considering their roles in the nervous system, information about their existence in the insect nervous system is rather sparse. Therefore, in order to detect monosaccharide content of N- and O-glycans, we carried out capLC–ESI–MS/MS analysis to determine the concentration changes of glucose, mannose, galactose, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose, xylose, arabinose, and ribose monosaccharides in the nervous system of Bombyx mori during development and aging processes. In addition to LC–MS, lectin blotting was done to detect quantitative changes in N- and O-glycans. Developmental stages were selected as 3rd (the youngest sample), 5th (young) larval instar, motionless prepupa (the oldest sample), and pupa (adult development). Derivatization of monosaccharides was performed with a solution of PMP agent and analyzed with capLC–ESI–MS/MS. For lectin blotting, determination of glycan types was carried out with Galanthus nivalis agglutinin and Peanut agglutinin lectins. In all stages, the most abundant monosaccharide was glucose. Although all monosaccharides were present most abundantly in the youngest stage (3rd instar), they are generally reduced gradually during the aging process. It was observed that amounts of monosaccharides increased again in the pupa stage. According to lectin blotting, N- and O-linked glycoproteins expressions were different and there were some specific glycoprotein expression differences between stages. These findings suggest that the glycosylation state of proteins in the nervous system changes during development and aging in insects in a similar fashion to that reported for vertebrates.     

Butyric acid production from red algae by a newly isolated <Emphasis Type="Italic">Clostridium</Emphasis> sp. S1

Objective

To produce butyric acid from red algae such as Gelidium amansii in which galactose is a main carbohydrate, microorganisms utilizing galactose and tolerating inhibitors in hydrolysis including levulinic acid and 5-hydroxymethylfurfural (HMF) are required.

Results

A newly isolated bacterium, Clostridium sp. S1 produced butyric acid not only from galactose as the sole carbon source but also from a mixture of galactose and glucose through simultaneous utilization. Notably, Clostridium sp. S1 produced butyric acid and a small amount of acetic acid with the butyrate:acetate ratio of 45.4:1 and it even converted acetate to butyric acid. Clostridium sp. S1 tolerated 0.5–2 g levulinic acid/l and recovered from HMF inhibition at 0.6–2.5 g/l, resulting in 85–92 % butyric acid concentration of the control culture. When acid-pretreated G. amansii hydrolysate was used, Clostridium sp. S1 produced 4.83 g butyric acid/l from 10 g galactose/l and 1 g glucose/l.

Conclusion

Clostridium sp. S1 produces butyric acid from red algae due to its characteristics in sugar utilization and tolerance to inhibitors, demonstrating its advantage as a red algae-utilizing microorganism.

     

Design and synthesis of cholecystokinin-4 dipeptide analogues with anxiolytic and anxiogenic activities

A new series of dipeptide analogues of the general formula Ph(CH₂) _n CO-NH(CH₂) _m CO-Trp-NH₂ (n = 1, 3–5; m = 1–3) was designed based on the structure of the endogenous tetrapeptide cholescystokinin-4 (CCK-4) and the topochemical Shemyakin-Ovchinnikov-Ivanov principle. The L-tryptophan derivatives exhibited anxiolytic properties and the D-tryptophan derivatives, anxiogenic properties. The dipeptide Ph(CH₂)₅CO-Gly-L-Trp-NH₂ (GB-115) with the activity in rats of 0.05–0.2 mg/kg after oral and intraperitoneal administration was chosen for further studies as a promising anxiolytic agent.     

Properties of Functional Films Based on Chitosan Derivative with Gallic Acid

Conjugates of chitosan (molecular weight 28 and 830 kDa) with gallic acid were synthesized by a reaction initiated by a free radical. The conjugates contained 106 and 119 mg of polyphenol per g of polymer. Thin films were obtained from solutions of synthesized chitosan derivatives, and their physico-chemical characteristics (thickness of 0.058–0.076 mm and moisture content of 7.92–9.44%), as well as the antioxidant (inhibiting activity with respect to 2,2-diphenyl-1-picrylhydrazyl) and antimicrobial properties, were studied in relation to Staphylococcus epidermidis and Escherichia coli.     

Effect of salt stress on the antimicrobial activity of <Emphasis Type="Italic">Ruta chalepensis</Emphasis> essential oils

In the current investigation, the biological activities of essential oils obtained from organs of Ruta chalepensis plants grown under salt stress (0, 50 and 100 mM NaCl) were analyzed. Their chemical composition was often investigated by GC/FID and GC–MS and the antimicrobial activities towards eight bacteria (Salmonella All, Salmonella K, Escherichia coli 45AG, Escherichia coli 45AI, Staphylococcus aureus 9402, Staphylococcus aureus 02B145, Listeria 477 and Pseudomonas aeruginosa ATCC 10145) and five fungi strains (Aspergillus, Saccharomycee crvisiale, Streptomyces griseus, Fusarium solani and Penicillium thomii) were studied. Results revealed that salt increased essential oil production in leaves at 50 and 100 mM NaCl. A total of 20 compounds were identified in leaves, undecan-2-one, nonan-2-one and geijerene being the dominant ones. In stems, 21 compounds were found; they were dominated by decan-2-one, geijerene, nonan-2-one and undecan-2-one. In contrast, roots exhibited a large variation with 25 volatile compounds and octyl acetate, methyl decanoate, phytyl acetate were the major ones. Salt stress induced significant antibacterial activity changes, mainly in leaves and stems. In leaves, the minimum inhibitory and bactericidal concentration decreased at 100 mM NaCl against Listeria 477, the two strains of E. coli (45AG and 45AI) and P. aeruginosa but it increased versus other bacteria. In stems, salt increased oil antibacterial activity against all strains except P. aeruginosa ATCC 10145. Root oil showed the least antibacterial activity under saline conditions versus Listeria 477 and P. aeruginosa ATCC 10145. As regards antifungal activity, NaCl reduced the antifungal activity of essential oils against the majority of fungi strains.     

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.