基于与木糖进行美拉德反应的低聚壳聚糖衍生物的抗氧化性能研究

研究低聚壳聚糖与木糖的美拉德反应,考察了两种体系(低聚壳聚糖与木糖的质量比分别为1∶1和1∶3)反应过程中pH、吸光度及荧光值的变化,醇沉法提取4 h和8 h的低聚壳聚糖美拉德反应衍生物,分别为CX11-4、CX13-4、CX11-8和CX13-8。对衍生物进行红外表征和分子量测定,并研究其对羟基自由基.OH和DPPH的清除能力以及还原能力。结果显示:壳聚糖衍生物的抗氧化能力都明显优于低聚壳聚糖,抗氧化活性顺序为CX13-4>CX11-4,CX11-8>CX13-8。可见,壳聚糖美拉德衍生物的抗氧化活性不仅与反应物的比例有关,还与反应的时间有关。     

Superoxide anion scavenging activity of graft chitosan derivatives

Two kinds of graft chitosan derivatives (CMCTS-g-MAS and HPCTS-g-MAS) were prepared by the graft copolymerization of maleic acid sodium onto etherified chitosans-carboxymethyl chitosan (CMCTS) and hydroxypropyl chitosan (HPCTS), respectively. Superoxide anion scavenging activity of the derivatives was evaluated in a luminal-enhanced autoxidaton of pyrogallol by chemiluminescence techniques. Compared with chitosan, the graft chitosan derivatives have much improved scavenging ability against superoxide anion. They have similar 50% inhibition concentrations (IC₅₀s) as ascorbic acid and superoxide dismutase (SOD). Graft chitosan derivatives with hydroxypropyl groups have relatively higher superoxide anion scavenging ability owing to the incorporation of hydroxyl groups. The graft chitosan derivatives (HPCTS-g-MAS 1, 2, and 3) with different grafting percentages exhibit IC₅₀s values ranging from 243 to 308 μg/mL, which could be related to the contents of active hydroxyl and amino groups in the polymer chains.     

Antioxidant properties of chitosan from crab shells

Crab chitosan was prepared by alkaline N-deacetylation of crab chitin for 60, 90 and 120 min and its antioxidant properties studied. Chitosan exhibited showed antioxidant activities of 58.3–70.2% at 1 mg/mL and showed reducing powers of 0.32–0.44 at 10 mg/mL. At 10 mg/mL, the scavenging ability of chitosan C60 on 1,1-diphenyl-2-picrylhydrazyl radicals was 28.4% whereas those of other chitosans were 46.4–52.3%. At 0.1 mg/mL, scavenging abilities on hydroxyl radicals were 62.3–77.6% whereas at 1 mg/mL, chelating abilities on ferrous ions were 82.9–96.5%. All EC₅₀ values of antioxidant activity were below 1.5 mg/mL. With regard to antioxidant properties assayed, the effectiveness of chitosans C60, C90 and C120 correlated with their N-deacetylation times. Overall, crab chitosan was good in antioxidant activity, scavenging ability on hydroxyl radicals and chelating abilities on ferrous ions and may be used as a source of antioxidants, as a possible food supplement or ingredient in the pharmaceutical industry.     

低聚壳聚糖与α-丙氨酸/天冬酰胺的美拉德反应及其衍生物的抗氧化性能研究

研究低聚壳聚糖(COS)与α-丙氨酸/天冬酰胺的美拉德反应,考察了两个体系(低聚壳聚糖的羰基与氨基的物质量比均为1∶1)的pH、吸光度和荧光值的变化。醇沉法提取低聚壳聚糖衍生物CA和CN。对两种衍生物进行红外表征和分子量测定,并研究其对超氧阳离子O2-.、DPPH自由基的清除能力以及还原能力。结果显示:抗氧化能力强弱次序为CA>CN>COS,即美拉德反应后低聚壳聚糖衍生物抗氧化能力得到显著提高,且CA的抗氧化活性优于CN,表明与小分子氨基酸进行美拉德反应制得的壳聚糖衍生物具有更好的抗氧化性。     

不同取代度N-邻苯二甲酰壳聚糖抗氧化活性的研究

低聚壳聚糖与邻苯二甲酸酐酰化得到三种取代度不同的N-邻苯二甲酸酐酰低聚壳聚糖NPCOSA、NPCOSB和NPCOSC,取代度分别为0.330、.55和0.65。通过红外光谱对其结构进行表征。并考察了其抗氧化性能。结果表明:COS的抗氧化性能最强;随着取代度的增加,N-邻苯二甲酰低聚壳聚糖对超氧阴离子的清除能力逐渐升高;而对DPPH的清除能力以及还原能力呈逐渐下降趋势;对羟基自由基的清除顺序大小依次为NPCOSB>NPCOSA>NPCOSC,即NPCOSB清除羟基自由基的的能力最佳。     

Preparation of 1,3,5-thiadiazine-2-thione derivatives of chitosan and their potential antioxidant activity in vitro

Three new kinds of 1,3,5-thiadiazine-2-thione derivatives of chitosan with two different molecular weight (SATTCS1, SATTCS2, TITTCS1, TITTCS2, CITTCS1 and CITTCS2) have been prepared. Their structures were characterized by IR spectroscopy. The substitution degree of derivatives calculated by elemental analyses was 0.47, 0.42, 0.41, 0.38, 0.41 and 0.36, respectively. The result shows that substitution degree of derivatives was higher with lower molecular weight. The antioxidant activity was studied using an established system, such as hydroxyl radical scavenging, superoxide radical scavenging and reducing power. Antioxidant activity of the 1,3,5-thiadiazine-2-thione derivatives of chitosan were stronger than that of chitosans and antioxidant activity of low molecular weight derivatives were stronger than that of high molecular weight derivatives. It is a potential antioxidant in vitro.     

Improvement of antioxidant activity of chitosan by chemical treatment and ionizing radiation

Modification of chitosan (CS) to N-maleoylchitosan (NMCS), N-phthaloylchitosan (NPhCS) and sulfonated-chitosan (SCS) was done using maleic anhydride, phthalic anhydride and chlorosulfonic acid, respectively followed by exposing them to γ-rays at different doses. The molecular weights and structural changes of irradiated chitosan derivatives were determined by GPC, FT-IR and UV-vis spectrophotometer. The molecular weights decreased with increasing irradiation dose. Results revealed that the main polysaccharide structure remained after irradiation. To investigate the enhancement of antioxidant activity of chitosan and its derivatives of different molecular weights, radical mediated lipid peroxidation inhibition, scavenging effect of DPPH radicals, reducing power and ferrous ion chelating activity assays were used. Chitosan derivatives with different molecular weights exhibit antioxidant activity. The lower the molecular weights of chitosan and its derivatives, the higher the antioxidant activity. NMCS possessed high scavenging effect on DPPH radicals compared with NPhCS, SCS and ascorbic acid. The irradiated chitosan and its derivatives could be used as natural antioxidants.     

The synthesis and antioxidant activity of the Schiff bases of chitosan and carboxymethyl chitosan

Five kinds of Schiff bases of chitosan and carboxymethyl chitosan (CMCTS) have been prepared according to a previous method and the antioxidant activity was studied using an established system, such as superoxide and hydroxyl radical scavenging. Obvious differences between the Schiff bases of chitosan and CMCTS were observed, which might be related to contents of the active hydroxyl and amino groups in the molecular chains.     

Factors affecting the free radical scavenging behavior of chitosan sulfate

Scavenging activity of hydroxyethyl chitosan sulfate (HCS) against 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl and carbon-centered radical species were studied using electron spin resonance (ESR) spectroscopy. In addition, its antioxidant activity to retard lipid peroxidation was also evaluated in a linoleic acid model system. HCS could scavenge DPPH (33.78%, 2.5 mg/mL) and carbon-centered radicals (67.74%, 0.25 mg/mL) effectively. However, chitosan sulfate did not exhibit any scavenging activity against hydroxyl radicals, but increased its generation. This was different from the published literature and was presumed due to the loss of chelating ability on Fe2+. This assumption could further confirm from the results obtained for Fe2+-ferrozine method that upon sulfation chitooligosaccharides lost its chelation properties. Therefore, HCS can be identified as antioxidant that effectively scavenges carbon centered radicals to retard lipid peroxidation.     

The preparation and antioxidant activity of the sulfanilamide derivatives of chitosan and chitosan sulfates

Chitosan (CS) and chitosan sulfates (CSS) with different molecular weight (Mw) were reacted with 4-acetamidobenzene sulfonyl chloride to obtain sulfanilamide derivatives of chitosan and chitosan sulfates (LSACS, HSACS, LSACSS, HSACSS). The preparation conditions such as different reaction time, temperature, solvent, and the molar ratio of reaction materials are discussed in this paper. Their structures were characterized by FTIR spectroscopy and elemental analyses. The antioxidant activities of the derivatives were investigated employing various established in vitro systems, such as hydroxyl-radical ((*)OH) superoxide anion (O2(*-)) scavenging and reducing power. All kinds of the compounds (HCS, LCS, HCSS, LCSS, HSACS, LSACS, HSACSS, LSACSS) showed stronger scavenging activity on hydroxyl radical than ascorbic acid (Vc). The inhibitory activities of the derivatives toward superoxide radical by the PMS-NADH system were obvious. The experiment showed that the superoxide radical scavenging effect of sulfanilamide derivatives of chitosan and chitosan sulfates was stronger than that of original CS and CSS. All of the derivatives were efficient in the reducing power. The results indicated that the sulfanilamide group were grafted on CS and CSS increased the reducing power of them obviously.     

Relevance of molecular weight of chitosan and its derivatives and their antioxidant activities in vitro

The antioxidant potency of different molecular weight (DMW) chitosan and sulfated chitosan derivatives was investigated employing various established in vitro systems, such as superoxide (O(2)(.-))/hydroxyl ((-.)OH) radicals scavenging, reducing power, iron ion chelating. As expected, we obtained several satisfying results, as follows: firstly, low molecular weight chitosan had stronger scavenging effect on O(2)(.-) and (-.)OH than high molecular weight chitosan. For example the O(2)(.-) scavenging activity of low molecular weight chitosan (9 kDa) and high molecular weight chitosan (760 kDa) were 85.86% and 35.50% at 1.6 mg/mL, respectively. Secondly, comparing with DMW chitosan, DMW sulfated chitosans had the stronger inhibition effect on O(2)(.-). At 0.05 mg/mL, the scavenging activity on O(2)(.-) reached 86.26% for low molecular weight chitosan sulfate (9 kDa), but that of low molecular weight chitosan (9 kDa) was 85.86% at 1.6 mg/mL. As concerning chitosan and sulfated chitosan of the same molecular weight, scavenging activities of sulfated chitosan on superoxide and hydroxyl radicals were more pronounced than that of chitosan. Thirdly, low molecular weight chitosan sulfate had more effective scavenging activity on O(2)(.-) and (-.)OH than that of high molecular weight chitosan sulfate. Fourthly, DMW chitosans and sulfated chitosans were efficient in the reducing power, especially LCTS. Their orders were found to be LCTS>CTS4>HCTS>CTS3>CTS2>CTS1>CTS. Fifthly, CTS4 showed more considerable ferrous ion-chelating potency than others. Finally, the scavenging rate and reducing power of DMW chitosan and sulfated derivatives increased with their increasing concentration. Moreover, change of DMW sulfated chitosans was the most pronounced within the experimental concentration. However, chelating effect of DMW chitosans were not concentration dependent except for CTS4 and CTS1.     

Antioxidant activity of differently regioselective chitosan sulfates in vitro

Differently regioselective chitosan sulfates were prepared according to Hanno Baumann's methods. Their antioxidant potencies were investigated employing various established in vitro systems, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH)/superoxide/hydroxyl radicals scavenging, reducing power, iron ion chelating and total antioxidant activity. All kinds of sulfated chitosans (HCTS, TSCTS, SCTS, TCTS) showed strong inhibitory activity toward superoxide radical by the PMS-NADH system compared to Vc. According to the above-mentioned order their IC50 were 0.012, 0.040, 0.015, 0.022 mg/mL, respectively, however, scavenging activity of Vc on superoxide radical was 68.19% at 2.0 mg/mL. Scavenging activity of superoxide radical was found to be in the order of HCTS>SCTS>TCTS>TSCTS>Vc. Furthermore, all kinds of sulfated chitosans exhibited strong concentration-dependent inhibition of deoxyribose oxidation. Except for HCTS, others had stronger scavenging activity on hydroxyl radical than Vc. Scavenging effect of TSCTS on 1,1-diphenyl-2-picrylhydrazyl radical was little lower than that of BHA, but better than that of others. All kinds of sulfated chitosans were efficient in the reducing power, especially TSCTS. TSCTS and TCTS showed considerable ferrous ion chelating potency. The data obtained in vitro models clearly establish the antioxidant potency of all kinds of sulfated chitosans. These in vitro results suggested the possibility that sulfated chitosans could be effectively employed as ingredient in health or functional food, to alleviate oxidative stress. However, comprehensive studies need to be conducted to ascertain the in vivo safety of sulfated chitosans in experimental animal models.     

Chitosan gallate as a novel potential polysaccharide antioxidant: an EPR study

A novel biopolymer-based antioxidant, chitosan conjugated with gallic acid (chitosan galloylate, chitosan-GA), is proposed. Electron paramagnetic resonance (EPR) demonstrates a wide range of antioxidant activity for chitosan-GA as evidenced from its reactions with oxidizing free radicals, that is, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), horseradish peroxidase (HRP)-H₂O₂, carbon-centered alkyl radicals, and hydroxyl radicals. The EPR spectrum of the radical formed on chitosan-GA was attributed to the semiquinone radical of the gallate moiety. The stoichiometry and effective concentration (EC₅₀) of the DPPH free radical with chitosan-GA show that the radical scavenging capacity is maintained even after thermal treatment at 100 °C for an hour. Although the degree of substitution of GA on chitosan was about 15%, its antioxidant capacity, that is, the reaction with carbon-centered and hydroxyl radicals, is comparable to that of GA.     

Antioxidant and antibacterial activities of eugenol and carvacrol‐grafted chitosan nanoparticles

Essential oils are known to possess antimicrobial and antioxidant activity while chitosan is a biocompatible polymer with antibacterial activity against a broad spectrum of bacteria. In this work, nanoparticles with both antioxidant and antibacterial properties were prepared by grafting eugenol and carvacrol (two components of essential oils) on chitosan nanoparticles. Aldehyde groups were first introduced in eugenol and carvacrol, and the grafting of these oils to chitosan nanoparticles was carried out via the Schiff base reaction. The surface concentration of the grafted essential oil components was determined by X‐ray photoelectron spectroscopy (XPS). The antioxidant activities of the carvacrol‐grafted chitosan nanoparticles (CHCA NPs) and the eugenol‐grafted chitosan nanoparticles (CHEU NPs) were assayed with diphenylpicrylhydrazyl (DPPH). Antibacterial assays were carried out with a representative gram‐negative bacterium, Escherichia coli (E. coli) and a gram‐positive bacterium, Staphylococcus aureus (S. aureus). The grafted eugenol and carvacrol conferred antioxidant activity to the chitosan nanoparticles, and the essential oil component‐grafted chitosan nanoparticles achieved an antibacterial activity equivalent to or better than that of the unmodified chitosan nanoparticles. Cytotoxicity assays using 3T3 mouse fibroblast showed that the cytotoxicity of CHEU NPs and CHCA NPs were significant lower than those of the pure essential oils. Biotechnol. Bioeng. 2009; 104: 30–39 © 2009 Wiley Periodicals, Inc.     

Quaternization of N-aryl chitosan derivatives: synthesis, characterization, and antibacterial activity

Chemical modification of chitosan by introducing quaternary ammonium moieties into the polymer backbone renders excellent antimicrobial activity to the adducts. In the present study, we have synthesized 17 derivatives of chitosan consisting of a variety of N-aryl substituents bearing either electron-donating or electron-withdrawing groups. Selective N-arylation of chitosan was performed via Schiff bases formed by the reaction between the 2-amino groups of the glucosamine residue of chitosan with aromatic aldehydes under acidic conditions, followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Each of the derivatives was further quaternized using N-(3-chloro-2-hydroxypropyl)trimethylammonium chloride (Quat-188) as the quaternizing agent that reacted with either the primary amino or hydroxyl groups of the glucosamine residue of chitosan. The resulting quaternized materials were water soluble at neutral pH. Minimum inhibitory concentration (MIC) antimicrobial studies of these materials were carried out on Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria in order to explore the impact of the extent of N-substitution (ES) on their biological activities. At ES less than 10%, the presence of the hydrophobic substituent, such as benzyl and thiophenylmethyl, yielded derivatives with lower MIC values than chitosan Quat-188. Derivatives with higher ES exhibited reduced antibacterial activity due to low quaternary ammonium moiety content. At the same degree of quaternization, all quaternized N-aryl chitosan derivatives bearing either electron-donating or electron-withdrawing substituents did not contribute antibacterial activity relative to chitosan Quat-188. Neither the functional group nor its orientation impacted the MIC values significantly.     

Reactive oxygen species scavenging activity of aminoderivatized chitosan with different degree of deacetylation

Chitosans with different degree of deacetylation were prepared from crab shell chitin in the presence of alkali. Aminoderivatized chitosan derivatives were prepared in addition of amino functional groups at a hydroxyl site in the chitosan backbone. Six kinds of aminoderivatized chitosan such as aminoethyl-chitosan (AEC90), dimethylaminoethyl-chitosan (DMAEC90), and diethylaminoethyl-chitosan (DEAEC90), which were prepared from 90% deacetylated chitosan, and AEC50, DMAEC50 and DEAEC50, which were prepared from 50% deacetylated chitosan, were prepared and their reactive oxygen species (ROS) scavenging activities were investigated against hydroxyl radical, superoxide anion radical and hydrogen peroxide. The electron spin resonance (ESR) spectrum revealed that AEC90 showed the highest scavenging effects against hydroxyl and superoxide anion radical, the effects were 91.67% and 65.34% at 0.25 and 5 mg/mL, respectively. For hydrogen peroxide scavenging effect, DEAEC90 exhibited the strongest activity. These results suggest that the scavenging effect depends on their degree of deacetylation and substituted group.     

Reactions of chitosan: 4. Preparation of organosoluble derivatives of chitosan

Fully subtituted di-O-acetyl-N-acetylchitosan (chitin diacetate) has been prepared by a route in which the hydroxyl groups are acetylated prior to N-acetylation. This overcomes the previously reported intramolecular steric hindrance to esterification caused by the N-acetamido group. The resultant products were of high viscosity but had a limited solubility range. Di-O-acrylcarbamate derivatives of N-acetylchitosan (chitin) have been produced by a similar route, whilst di-O-arylcarbamate-N-arylureidochitosans have been prepared directly from chitosan. These products also have limited solubility ranges and have inherent viscosities similar to that of di-O-acetylchitosan prepared from the same batch of chitosan.     

Antifungal property of quaternized chitosan and its derivatives

Five water-soluble chitosan derivatives were carried out by quaternizing either iodomethane or N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride (Quat188) as a quaternizing agent under basic condition. The degree of quaternization (DQ) ranged between 28 ± 2% and 90 ± 2%. The antifungal activity was evaluated by using disc diffusion method, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) methods against Trichophyton rubrum (T. rubrum), Trichophyton mentagrophyte (T. mentagrophyte), and Microsporum gypseum (M. gypseum) at pH 7.2. All quaternized chitosans and its derivatives showed more effective against T. rubrum than M. gypseum and T. mentagrophyte. The MIC and MFC values were found to range between 125-1000 μg/mL and 500-4000 μg/mL, respectively against all fungi. Our results indicated that the quaternized N-(4-N,N-dimethylaminocinnamyl) chitosan chloride showed highest antifungal activity against T. rubrum and M. gypseum compared to other quaternized chitosan derivatives. The antifungal activity tended to increase with an increase in molecular weight, degree of quaternization and hydrophobic moiety against T. rubrum. However, the antifungal activity was depended on type of fungal as well as chemical structure of the quaternized chitosan derivatives.     

Synthesis, characterization, and antifungal activity of novel quaternary chitosan derivatives

Three novel quaternary chitosan derivatives were successfully synthesized by reaction of chloracetyl chitosan (CACS) with pyridine (PACS), 4-(5-chloro-2-hydroxybenzylideneamino)-pyridine (CHPACS), and 4-(5-bromo-2-hydroxybenzylideneamino)-pyridine (BHPACS). The chemical structure of the prepared chitosan derivatives was confirmed by Fourier transform infrared (FT-IR) and ¹³C nuclear magnetic resonance (¹³C NMR) and their antifungal activity against Cladosporium cucumerinum, Monilinia fructicola, Colletotrichum lagenarium, and Fusarium oxysporum was assessed. Comparing with the antifungal activity of chitosan, CACS, and PACS, CHPACS and BHPACS exhibited obviously better inhibitory effects, which should be related to the synergistic reaction of chitosan itself with the grafted 2-[4-(5-chloro-2-hydroxybenzylideneamino)-pyridyl]acetyl and 2-[4-(5-bromo-2-hydroxybenzylideneamino)-pyridyl]acetyl.     

Sulfation of a polysaccharide produced by a marine filamentous fungus Phoma herbarum YS4108 alters its antioxidant properties in vitro

Free radicals and other reactive oxygen species (ROS) are generated by all aerobic cells and are widely believed to play a significant role in aging as well as a number of degenerative or pathological diseases. This study compared the free radical-scavenging properties and antioxidant activity of YCP, a polysaccharide from the mycelium of a marine filamentous fungus Phoma herbarum YS 4108 and its two chemically sulfated derivatives YCP-S1 and YCP-S2. Sulfation, which masks hydroxyl groups of YCP polysaccharide molecule, could introduce new antioxidant activity, such as superoxide and hydroxyl radicals scavenging activity, metal chelating action, lipid peroxidation and linoleic acid oxidation inhibition capability. Furthermore, sulfated YCP was more potent than YCP at protecting erythrocytes against oxidative damage hemolysis. The current data suggest for the first time that sulfation of polysaccharide significantly increases its antioxidant activity and the chemical modification of polysaccharides may allow the preparation of derivatives with new properties and a variety of applications.