Preparation and antimicrobial activity of hydroxypropyl chitosan

Water-soluble hydroxypropyl chitosan (HPCS) derivatives with different degrees of substitution (DS) and weight-average molecular weight (Mw) were synthesized from chitosan and propylene epoxide under basic conditions. Their structure was characterized by IR spectroscopy, NMR spectroscopy, and elemental analysis, which showed that both the OH groups at C-6 and C-3 and the NH2 group of chitosan were alkylated. The DS value of HPCS ranged from 1.5 to 3.1 and the Mw was between 2.1x10(4) and 9.2x10(4). In vitro antimicrobial activities of the HPCS derivatives were evaluated by the Kirby-Bauer disc diffusion method and the macrotube dilution broth method. The HPCS derivatives exhibited no inhibitory effect on two bacterial strains (Escherichia coli and Staphylococcus aureus); however, some inhibitory effect was found against four of the six pathogenic fruit fungi investigated. Some derivatives (HPCS1, HPCS2, HPCS3, HPCS3-1, and HPCS4) were effective against C. diplodiella and F. oxysporum. HPCS3-1 is the most effective one with MIC values of 5.0, 0.31, 0.31, and 0.16mg/mL against A. mali, C. diplodiella, F. oxysporum, and P. piricola, respectively. Antifungal effects were also observed for HPCS2 and HPCS3-1 against A. mali, as well as HPCS3 and HPCS3-1 against P. piricola. The results suggest that relatively lower DS and higher Mw value enhances the antifungal activity of HPCS derivatives.     

Effects of concentration, degree of deacetylation and molecular weight on emulsifying properties of chitosan

Chitosan has excellent emulsifying properties. Emulsifying activity and stability of chitosan were determined by integrated light scattering technique and turbidimetric method. The effects of concentration, degree of deacetylation and molecular weight on emulsifying properties of chitosan were systematically studied in the paper. Emulsifying activity of chitosan initially increased, arrived at the peak at 0.75% and then declined, while emulsifying stability continuously increased with a rise of chitosan concentration from 0.25% to 1.25%. Emulsifying activity and stability of chitosan initially decreased and reached the minimum, then increased with the rise of degree of deacetylation. Chitosan with DD 60.5% and 86.1% showed superior emulsifying activity and stability. Chitosan with low Mw exhibited better emulsifying activity than those with high Mw. Chitosan with Mw 410 kDa and 600 kDa showed superior emulsifying activity in the test range. Emulsifying stability of chitosan increased with a rise of Mw.     

Studies on the antibacterial activities and mechanisms of chitosan obtained from cuticles of housefly larvae

Chitosan was obtained from cuticles of the housefly (Musca domestica) larvae. Antibacterial activities of different Mw chitosans were examined against six bacteria. Antibacterial mechanisms of chitosan were investigated by measuring permeability of bacterial cell membranes and observing integrity of bacterial cells. Results show that the antibacterial activity of chitosan decreased with increase in Mw. Chitosan showed higher antibacterial activity at low pH. Ca2+ and Mg2+ could markedly reduce the antibacterial activity of chitosan. The minimum inhibitory concentrations of chitosans ranged from 0.03% - 0.25% and varied with the type of bacteria and Mw of chitosan. Chitosan could cause leakage of cell contents of the bacteria and disrupt the cell wall.     

Antitumor activity of levan polysaccharides from selected microorganisms

Levans were isolated from the cultures of Gluconoacetobacter xylinus (G-levan; Mw = 40,000), Microbacterium laevaniformans (M; Mw = 710,000), Rahnella aquatilis (R; Mw = 380,000), and Zymomonas mobilis (Z; Mw = 570,000). The levans were composed mainly of fructose residues when analyzed by TLC and HPLC, and their main backbones were beta-(2,6)-linkages with beta-(2,1)-branches by GC-MS and NMR. In the in vitro antitumor activity test of the levans against eight different tumor cell lines, relatively stronger activity was observed from the SNU-1 and HepG2. The M- (52.54-62.05%) and R-levan (52.15-58.58%) showed the significantly high activity against SNU-1, while M-levan showed the highest (49.93-61.82%) activity against HepG2. During the in vivo analysis of inhibitory activity of the levans against Sarcoma-180 growth, M-, R- and Z-levans showed strong antitumor activity (average 66%) but G-levan (42%) had significantly lower activity.     

温敏型壳聚糖介入核素188Re内照射抗小鼠移植性肝癌（H22）

目的研究温敏型壳聚糖（chitonsan CS）介入核素188Re内照射对小鼠移植性肝癌（H22）的抑制作用。方法建立小鼠肝癌（H22）模型后随机分成7组,即模型对照组、188Re（0.1mCi）组、188Re-S（0.1mCi）组、188Re＋CS（0.1mCi）组、188 Re＋CS（0.2mCi）组、188 Re＋硫胶体＋壳聚糖（188 Re-S＋CS 0.1mCi）组和188 Re-S＋CS（0.2mCi）组。各组动物瘤内分别注射相应试药,测定肿瘤抑制率。结果 188Re＋CS组和188Re-S＋CS组肿瘤生长速度减慢,肿瘤生长延迟,肿瘤抑制率在治疗后6 d最高,抑制率分别为67.35%和67.81%。结论温敏型壳聚糖介入核素188Re内照射对小鼠肝癌（H22）具有一定的抑制作用。     

Correlation between the antitumor activity of a polysaccharide schizophyllan and its triple-helical conformation in dilute aqueous solution

Eight samples of a polysaccharide schizophyllan ranging in weight-average molecular weight Mw (in water) from 5 x 10(3) to 1.3 x 10(5) were prepared and their antitumor activity (expressed in terms of the tumor inhibition ratio) against Sarcoma 180 ascites, intrinsic viscosities [eta], and gel-filtration chromatograms in aqueous solution were determined. The tumor inhibition ratio was essentially unity for samples with Mw higher than 9 x 10(4), but reduced to zero or even to a negative value when Mw was lower than 10(4). The [eta] data combined with the chromatographic data showed that above Mw approximately 9 x 10(4) the predominant species of schizophyllan in aqueous solution is the previously found rigid triple helix, whereas below Mw approximately 9 x 10(4) both triple helices and single chains coexist in the solution and the fraction of triple helices decreases monotonically to zero as Mw is decreased to 5 x 10(3). From these findings it was concluded that the antitumor potency of schizophyllan in water is related to the amount of triple helices relative to that of single chains.     

The contribution of acidulant to the antibacterial activity of acid soluble α- and β-chitosan solutions and their films

This study evaluated individual contributions of dissolving acids (acetic acid, lactic acid, and hydrochloric acid) or acid solubilized chitosan to the antibacterial activity against Listeria innocua and Escherichia coli as solutions and dried films. Solutions containing chitosan showed significantly (P?<?0.05) different inhibitory activity (measured as percentage of inhibition (PI), in percent) against L. innocua and E. coli, compared to equivalent acid solutions. This increase was calculated as additional inhibition (AI, in percent), which could be as high as 65 % in solutions containing 300–320 kDa chitosan depending on the acid type, bacterial species, and the chitosan form (α or β). Solutions containing 4–5 kDa chitosan had lower AI and showed much greater variability among the different chitosan forms, acid types, and bacterial species. Higher molecular weight (Mw) chitosan also showed significantly higher levels of adsorption to bacterial cells than that of lower Mw samples, suggesting that the observed increase in inhibition was the result of surface phenomena. The contribution of acids to the antibacterial activity of chitosan films was assessed by comparing non-rinsed and rinsed films (rinsed in the appropriate broth to remove residual acids and active fragments formed on the dried film). Rinsing β-chitosan films has reduced PI by as much as 28 % compared with non-rinsed films, indicating that part of the antibacterial activity of chitosan films is due to the presence of soluble acid compounds and/or other active fragments. Overall, both acidulant and chitosan were found to contribute to the antibacterial activity of acid solubilized α- and β-chitosan, with the exact antibacterial activity of chitosan varying based on the solution and film properties, suggesting a complex interaction.     

Effect of molecular weight of chitosan on antimicrobial properties and tissue compatibility of chitosan-impregnated bacterial cellulose films

Bacterial cellulose-chitosan (BC-C) films were developed by immersing purified BC pellicles in 1.5 ~ 2.0% (w/v) acetic acid solutions containing chitosan of varying molecular weights. Effects of different molecular weight of chitosan on physical, biological and antimicrobial properties of the composite films were investigated. The cumulative chitosan absorption capacities with M_w of 141,000, 199,000, and 263,000 were 38.43, 24.65, and 23.89 mg/cm³ of dry BC film, respectively. The cumulative release profiles of chitosan from the films strongly depended on molecular weight of chitosan and pH of solution. The order of release of chitosan from the BC-C films was dependent on molecular weight as follows: M_w 141,000 > M_w 199,000 > M_w 263,000. All BC-C films showed the antimicrobial abilities against Staphylococcus aureus and Aspergillus niger but had no inhibitory effect on the growth of Escherichia coli. The BC-C films supported for adhesion, spreading and proliferation of both human skin keratinocytes and fibroblasts. The antibacterial activity against S. aureus of the BC-C with the highest Mw chitosan (263,000) was higher than those of the others. On the other hand, the BC-C films with the lowest M_w chitosan (141,000) promoted the growth of human skin cells more than those of the others.     

Comparison in antioxidant action between α-chitosan and β-chitosan at a wide range of molecular weight and chitosan concentration

Antioxidant activity in α- and β-chitosan at a wide range of molecular weight (Mw) and chitosan concentration (CS) was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing ability, chelating ability, and hydroxyl radical scavenging activity. The form of chitosan (FC) had significant (P <0.05) effect on all measurements except DPPH radical scavenging activity, and antioxidant activity was dependent on Mw and CS. High Mw (280–300 kDa) of β-chitosan had extremely lower half maximal effective concentrations (EC₅₀) than α-chitosan in DPPH radical scavenging activity and reducing ability. The 22–30 kDa of α- and β-chitosan showed significantly (P <0.05) higher activities in DPPH radical scavenging, reducing ability, and hydroxyl radical scavenging than samples at other Mw, while chelating ability was the highest in 4–5 kDa chitosan. CS had significant effect on all measurements and the effect was related to Mw. The antioxidant activity of 280–300 kDa chitosan was affected by coil-overlap concentrations (C¹) in the CS range of 4–10 mg/mL, forming entanglements. Reducing ability and hydroxyl radical scavenging activity were more predominant action in antioxidant activity of chitosan as shown by the lower EC₅₀ values than those in other antioxidant measurements.     

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.     

The preparation of low-molecular-weight chitosan using chitinolytic complex from Streptomyces kurssanovii

Streptomyces kurssanovii are Gram-positive mycelial bacteria ubiquitous in soil. They have a saprophytic way of life and produce many extracellular enzymes with polymer-degrading properties, for example, chitinase (EC 3.2.1.14) and N-acetyl-β- -glucosaminidase (EC3.2.1.30). Biochemical aspects of chitosan degradation were presented. Low-molecular-weight (LMW) chitosans with molecular weight 4–8 kDa were prepared from commercial crab chitosan by means of chitinolytic a complex from S. kurssanovii. The optimum conditions of process in solution (temperature, pH, enzyme-substrate ratio) have been determined. Yields of LMW chitosan were 70–80%.     

Rheological, water uptake and controlled release properties of a novel self-gelling aldehyde functionalized chitosan

In this work, aldehyde-functionalized chitosan was used to prepare gels through Schiff base formation between the aldehyde and the free amine groups. The gels, which were formed in situ without an external crosslinker, were characterized in respect to their rheological properties and water uptake capacity. In addition, the potential use of these gels as drug carriers was investigated using metronidazole (Mw 171Da), FTIC-dextran (Mw 40kDa) and bovine serum albumin (Mw 66kDa), as the model drugs. Higher concentrations of the aldehyde-functionalized chitosan originated more rigid gels, as evidenced by the higher values of complex modulus (G*). The highly porous structure of these gels allowed for more than 400% of water uptake. The release of the model drugs followed a near zero-order release profile. The molecular size as well as the pH of the medium influenced the amount of drug released, where the higher the pH, the slower the resulting drug release.     

Correlation of structure to antitumor activities of five derivatives of a beta-glucan from Poria cocos sclerotium

A water-insoluble (1-->3)-beta-D-glucan isolated from fresh sclerotium of Poria cocos was, respectively, sulfated, carboxymethylated, methylated, hydroxyethylated, and hydroxypropylated, to afford five water-soluble derivatives. Their weight-average molecular masses (Mw) and intrinsic viscosities ([eta]) were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The antitumor activities, against Sarcoma 180 tumor cell (S-180) and gastric carcinoma cell strain (MKN-45 and SGC-7901) of the native beta-glucan and the five derivatives, were tested in vitro and in vivo. The Mw values of the five derivatives in PBS were determined to be 3.8 x 10(4), 18.9 x 10(4), 16.0 x 10(4), 76.8 x 10(4), and 224.3 x 10(4), respectively. The high Mw values of the hydroxyethylated and hydroxypropylated derivatives in aqueous solution resulted from aggregation, and their true Mw values obtained in dimethyl sulfoxide were 20.1 x 10(4) and 19.1 x 10(4). The sulfated and carboxymethylated derivatives having DS of 1.0-1.3 show good water solubility, and exist as relatively expanded chains in aqueous solution. Interestingly, the native beta-glucan did not show antitumor activity, whereas the sulfated and carboxymethylated derivatives exhibit significant antitumor activities against S-180 and gastric carcinoma tumor cells. This work showed that good water solubility, relatively high chain stiffness, and moderate molecular mass of the derivatives in aqueous solution contribute beneficial to enhancement of antitumor activity.     

Kinin release from kininogens by calpains

During the investigation of inhibitory activity of kininogens toward calpains [EC 3.4.22.17], we found that lysyl-bradykinin was liberated from both high molecular weight (HMW) and low molecular weight (LMW) kininogens by the action of the calpains. The kinin liberation occurred in a limited range of calpain to kininogen molar ratios of 0.5:1 to 8:1, and in that condition calpains were simultaneously inhibited 20 to 80% by kininogens. The maximum level of kinin release from HMW and LMW kininogens by calpain I was about 25% and that by calpain II was 20%. These results suggest that in case of inflammation the kininogens play two physiologically distinct roles by interaction with calpains: to release lysyl-bradykinin and to inhibit proteinase activity of calpains derived from the damaged tissues.     

Evaluation of sulfated Lentinus edodes alpha-(1-->3)-D-glucan as a potential antitumor agent

Alpha-glucan L-FV-II and beta-glucan L-FV-I were shown to co-exist in the extract of fruiting bodies of Lentinus edodes with aq. 5% NaOH/0.05% NaBH4 in previous work. Water-insoluble alpha-(1-->3)-D-glucan (L-FV-II) was treated with sulfur trioxide-pyridine complex at 25 degrees C to synthesize the water-soluble sulfated derivative SL-FV-II with a degree of substitution (DS) 1.1 in non-selective sulfation. The weight-average molecular weight (Mw) of sulfated glucan SL-FV-II is 57% of that of the original alpha-glucan L-FV-II. The alpha-glucan administered by gavaging at a dose of 50 mg/kg of body weight to BALB/C mice having implanted solid Sarcoma 180 was effective at an inhibition rate of 42%. In vitro experiments using human and murine tumor cell lines showed that SL-FV-II had antiproliferation activity at the concentration of 20 microg/mL towards four tumor cell lines. The sulfated alpha-(1-->3)-D-glucan had potent antiproliferation action (52%) on human MCF-7 breast carcinoma cells.     

Biochemical activities of low molecular weight chitosans derived from squid pens

Chitosans were prepared by H₂O₂ oxidative depolymerization from squid pens with low molecular weights (LMW) of 13,025, 7011, 4169, 2242 and 963 Da. The bile acid binding capacities and antioxidant properties of LMW chitosans were studied in vitro. LMW chitosans exhibited stronger bile acid binding capacities than that of chitosan. The scavenging ability of LMW chitosans against DPPH radicals improved with increasing concentration, and EC₅₀ values were below 1.3 mg/mL. The EC₅₀ values of LMW chitosans against hydroxyl radicals ranged from 0.93 to 3.66 mg/mL. All LMW chitosans exhibited a strong ferrous ion chelating effect and reducing power. At 1 mg/mL, the scavenging ability of chitosan-963 towards superoxide radicals was 67.76%. These results indicated that LMW chitosans which have stronger bile acid binding capacity and antioxidant activities may act as potential antioxidants in vitro.     

Reclamation of chitinous materials by bromelain for the preparation of antitumor and antifungal materials

The main purpose of this research was to investigate the antitumor and antimicrobial activities of the chitooligosaccharides containing hydrolyzates obtained from the hydrolysis of chitinous materials (such as chitin, chitosan, and squid pen) by bromelain. The optimum preparations were gained in the hydrolysates of squid pen powder hydrolyzed at pH 5, 37 degrees C for 2 days by 0.1% bromelain. The hydrolysates had an 80% inhibitory activity on phyto-pathogenic mold Fusarium oxysporum. Chitooligosaccharides were recovered from the hydrolysates and were used for tumor cell surviving test. Surviving rate of the human leukemic U937 cells was reduced to 69% by the chitooligosaccharides. The solution of 0.1% of water-soluble chitosan was also hydrolyzed for 1 day at pH 5, 37 degrees C by bromelain. The resultant hydrolysates contained the highest chitooligosaccharides, which had inhibitory effect on Bacillus subtilis and also had 40% inhibitory activity on human pathogenic mold Aspergillus fumigatus. Surviving rate of mouse CT26 colorectal adenocarcinoma cells was reduced to 57% by the chitooligosaccharides. This is the first publication of enzymatic reclamation of squid pen (fishery processing waste) for the preparation of antitumor and antimicrobial materials.     

Glutaraldehyde cross-linked chitosan microspheres for controlled release of centchroman

Glutaraldehyde cross-linked chitosan microspheres were prepared for controlled release of centchroman, a nonsteroidal contraceptive. The cross-linked microspheres with low-molecular-weight (LMW) chitosan (260 kg mol(-1)) have shown maximum degree of swelling (287 wt%) but were found to be poor in loading and release behavior for centchroman. The microspheres with medium-molecular-weight (MMW) chitosan (1134 kg mol(-1)) have shown 250 wt% degree of swelling and 37.5 wt% loading of centchroman, but microspheres with high-molecular-weight (HMW) chitosan (2224 kg mol(-1)) have shown a low degree of swelling (150 wt%) and centchroman loading (30 wt%). The microspheres with MMW chitosan have released 82 wt% of loaded centchroman in a controlled release manner within a period of 70 h in comparison to low- (260 kg mol(-1)) and high-MW (2224 kg mol(-1)) chitosan microspheres. The chitosan microspheres with 62 wt% degree of deacetylation (DDA) were more efficient in the controlled release of centchroman in comparison to chitosan microspheres with low (48 wt%) and high-DDA (75 wt%). The fractional release of centchroman (M(t)/M(infinity)) from chitosan microspheres was used to predict the mechanism of drug release and to determine the diffusion constant (D) of centchroman.     

Molecular weight and pH effects of aminoethyl modified chitosan on antibacterial activity in vitro

Aminoethyl modified chitosan derivatives (AEMCSs) with different molecular weight (Mw) were synthesized by grafting aminoethyl group on different molecular weight chitosans and chitooligosaccharide. FTIR, (1)H NMR, (13)C NMR, elemental analysis and potentiometric titration results showed that branched polyethylimine chitosan was synthesized. Clinical Laboratory Standard Institute (CLSI) protocols were used to determine MIC for Gram-negative strain of Escherichia coli under different pH. The antibacterial activity of the derivatives was significantly improved compared with original chitosans, with MIC values against E. coli varying from 4 to 64 μg/mL depending on different Mw and pH. High molecular weight seems to be in favor of stronger antibacterial activity. At pH 7.4, derivatives with Mw above 27 kDa exhibited equivalent antibacterial activity (16 μg/mL), while oligosaccharide chitosan derivative with lower Mw (~1.4 kDa) showed decreased MIC of 64 μg/mL. The effect of pH on antibacterial activity is more complicated. An optimal pH for HAEMCS was found around 6.5 to give MIC as low as 4 μg/mL, while higher or lower pH compromised the activity. Cell integrity assay and SEM images showed evident cell disruption, indicating membrane disruption may be one possible mechanism for antibacterial activity.     

Controlling chitosan’s molecular weight via multistage deacetylation

Chitosan samples with different molecular weights (Mw) and degree of deacetylation (DD) were prepared by controlling operating conditions throughout the multistage alkali treatment. The temperature of the reaction, time duration and number of reaction steps were considered effective parameters. A database was developed for chitosan preparation in order to achieve high degrees of deacetylation and control the molecular weight of chitosan without changing other molecular structures. The number of treatments and the duration of each step of deacetylation significantly affected molecular weight so that two samples were obtained with a DD of 99% and two different molecular weights ranging from 4.66×10⁵ to 2.93×10⁵ Based on these results, the highest molecular weight obtained using the multistage treatment without decreasing DD was 5.32×10⁵, with a DD of 96.67%. Also, the morphological studies indicate that the molecular weight of chitosan has a significant effect on the pore size of the prepared scaffolds. However, this effect is critical. In other words, the pore size will increase by increasing molecular weight of chitosan from low upto medium molecular weight and when it reached to high molecular weight the pore size is decreased.