Monomer composition of chitooligosaccharides obtained by different degradation methods and their effects on immunomodulatory activities

Accepted Manuscript Title: Monomer composition of chitooligosaccharides obtained by different degradation methods and their effects on immunomodulatory activities Author: Ronge Xing Yongliang Liu Kecheng Li Huahua Yu Song Liu Yue Yang Xiaolin Chen Pengcheng Li PII: DOI: Reference:

S0144-8617(16)31257-7 http://dx.doi.org/doi:10.1016/j.carbpol.2016.11.001 CARP 11711

To appear in: Received date: Revised date: Accepted date:

19-8-2016 21-10-2016 2-11-2016

Please cite this article as: Xing, Ronge., Liu, Yongliang., Li, Kecheng., Yu, Huahua., Liu, Song., Yang, Yue., Chen, Xiaolin., & Li, Pengcheng., Monomer composition of chitooligosaccharides obtained by different degradation methods and their effects on immunomodulatory activities.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.11.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Monomer composition of chitooligosaccharides obtained by different degradation methods and their effects on immunomodulatory activities 1,2 Ronge Xing , Yongliang Liu1,2,Kecheng Li1,2, Huahua Yu1,2, Song Liu1,2, Yue Yang1,2, Xiaolin Chen1,2, Pengcheng Li1,2* 1 Institute of Oceanology, Chinese Academy of Sciences; 2 Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences  Corresponding author. Tel.:+86 532 82898707; fax: +86 532 82898780. E-mail address: [email protected]; [email protected] (P.L.)

Highlights  Three kinds of chitooligosaccharides were prepared through different methods.    Their monomer compositions were researched by GPC, MS and elemental analysis.  Oligosaccharides by different methods could significantly stimulate the immune system  WCOS obtained by microwave irradiation had the best immunomodulatory activities.  Activity of WCOS might relate to hexamer and acetyl group in molecule composition.    Abstract: Three kinds of chitooligosaccharides were prepared using traditional methods (ZCOS), microwave irradiation (WCOS), and enzymatic hydrolysis (YCOS), and their monomer compositions and in vitro and in vivo immunomodulatory activities were determined. ZCOS was mainly comprised of disaccharides and trisaccharides; WCOS was mainly comprised of disaccharides to hexasaccharides with or without acetyl groups; and YCOS was mainly comprised of trisaccharides to pentasaccharides without acetyl groups. Differences in monomer composition and acetyl groups affected their immunomodulatory activities. Chitooligosaccharides degraded by different methods all significantly stimulated the immune system by acting through cellular and humoral immunities. WCOS, obtained by microwave irradiation, had the best immunomodulatory activity. It significantly increased the spleen index and significantly stimulated delayed-type hypersensitivity compared the other two chitooligosaccharides. These activities might have been the result of the hexamer and acetyl groups of WCOS. A list of abbreviations: Chitooligosaccharides (COS), glucosamine (GlcN), Chitosan (CTS), Chitooligosaccharide by degraded enzyme method (YCOS), Chitooligosaccharide by degraded traditional methods (ZCOS), Chitooligosaccharide by degraded microwave irradiation (WCOS), 3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetrazolium bromide (MTT), Concanavalin A (ConA), lipopolysaccharide (LPS), and dimethyl sulphoxide (DMSO), fetal calf serum (FCS), high  performance liquid phase chromatography (HPLC), gel permeation chromatography (GPC), phosphate-buffered saline (PBS), natural killer cells (NK), lactate dehydrogenase (LDH), effector cells (YAC-1), delayed type hypersensitivity (DTH), mononuclear phagocytic system (MPS), cytotoxic T lymphocytes (CTLs), major histocompatibility complex (MHC). Keywords: Chitooligosaccharide; Degradation methods; Monomer composition; Immunomodulatory activities; Chitosan. Introduction: 1   

It is well known that immune function is important in the prevention and control of tumours, and many studies have reported that natural substances can prevent and cure neoplastic diseases through immunomodulation. Polysaccharides in many natural substances have been reported to significantly increase the immune response [Lim et al., 2004; Zhu and Chen, 2007]. These observations have resulted in great interest because polysaccharides are relatively nontoxic and have no side effects. Chitosan is a natural and nontoxic biopolysaccharide produced by the deacetylation of chitin, which is a major component of crustacean shells from crab, shrimp, and crawfish. Because of their special properties of biocompatibility, biodegradability, bioactivity, and nontoxicity, many studies have reported various applications of chitosan in agriculture, food, biomaterials, and medicine [Khor and Lim, 2003; Muzzarelli, 2010]. However, high viscosity in acid solutions and poor solubility at neutral pH restrict its applications. In addition, the molecular composition and molecular weight of chitosan have been reported to influence its properties and functions [No et al., 2002; Huang et al., 2004; Xing et al., 2005]. Chitooligosaccharides (COS) are hydrolysates of chitosan, mainly composed of 1,4-linked D-glucosamine (GlcN) and partially composed of 1,4-linked N-acetyl-D-glucosamine. The chemical structure of chitosan does not change in COS, and it exhibits better water solubility and greater physiological activities involving antitumor activity, free radical scavenging activity, antimicrobial activity, immune modulatory effects, and wound healing effects [Aam et al., 2010; Xia et al., 2011]. COS is currently produced by acidic hydrolysis, oxidative degradation, microwave irradiation, or enzymatic hydrolysis [Chang et al., 2001; Xing et al., 2005; Trombotto et al., 2008; Heggset et al., 2010]. All these methods of COS preparation produce mixtures of COS containing different molecular weights and different degrees of acetylation. COS with similar molecular weights obtained by different methods of degradation have different molecular compositions, containing different amounts of monomers. These differences result in different levels of the same activity. For example, Chen et al. found that chitobiose and chitotriose were more potent than glucosamine hydrochloride in scavenging hydroxyl radicals, and both had the ability to protect against CCl4-induced acute hepatoxicity [No et al., 2002; Huang et al, 2004;]. Chitohexaose and hexa-N-acetylchitohexaose have been reported to possess strong antitumor activities [Suzuki and Mikami, 1986; Xiong and Wu, 2009]. Chitooligosaccharides mainly consisting of chitohexaose have been reported to have a stimulatory effect on the release of interleukin-1β and tumour necrosis factor-α in macrophages in vitro [Feng, Zhao, and Yu, 2004]. Based upon these observations, three kinds of COS obtained by traditional methods, microwave irradiation, or enzymatic hydrolysis were prepared and their immunomodulatory activities were determined. In order to understand the effects of degradation methods on the chemical structure, composition, and biological activities of chitooligosaccharides, the COS produced by degradation using different methods was characterized by gel permeation chromatography, mass spectrometry, elemental analysis, and solubility studies. The immunomodulatory activities of COS obtained using the different methods were determined using in vitro and in vivo models.   2. Materials and methods 2.1. Materials, reagents, equipment and experiments

Chitosan from crab shell, whose degree of deacetylation and molecular weight 2   

were 82% and 658×103 Da, respectively, was purchased from Qingdao Yunzhou Biochemical Corp. (Qingdao, China). Chitooligosaccharide (YCOS) by degraded enzyme method, whose molecular weight was 1110Da by gel permeation chromatography (GPC) analysis, was purchased from Qingdao Yunzhou Biochemical Corp. (China). Glacial acetic acid, hydrogen peroxide (concentration 30%), sodium hydroxide, anhydrous alcohol and other chemicals were of analytical grade. 3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetrazolium bromide (MTT), Concanavalin A (ConA), lipopolysaccharide (LPS), and dimethyl sulphoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA).; PRMI-1640 medium and fetal calf serum (FCS) were from Gibco (Grand Island, NY, USA). Microwave synthesis/extraction reaction station (Type: MAS-II) was purchased from Shanghai SINEO Microwave Chemistry Technology Co., Ltd; A Vario EL-III elemental analyzer was bought from elementar Analysensysteme GmbH; An Agilent 1260 series HPLC system was purchased from Agilent Technologies Co., Ltd; a LTQ ORBITRAR XL mass spectrometer was purchased from Amecican Thermo Scientific; Incubator was purchased from Japanese SANYO corporation; Super-clean bench was purchased from Beijing East Harbin manufacturing company; Mouse electronic scale was purchased from Changshou City days instrument limited liability company; Inverted microscope was purchased from Olympus Co., LTD.; 1/10000 balance was purchased from Mettler Toledo; ELISA instrument was purchased from Tecan group Austria Co., Ltd.   2.2 Methods 2.2.1 Degradation of chitosan under microwave irradiation The chitosan powder (3g) was introduced in an Erlenmeyer flask containing 2% acetic acid (100mL, that is 2mL glacial acetic acid plus 98mL distilled water). Then hydrogen peroxide (H2O2, whose final concentration is 1%) was added to the chitosan aqueous solution. The mixture solution was heated for degradation of chitosan by microwave irradiation. The degradation with microwave irradiation was performed in a laboratory microwave reaction station with 1000W of power (Sineo Microwave Chemical Technology Co. Ltd., Shanghai, China) with a magnetic stirrer and an infrared reaction thermometer. The experiments were employed with the power of 800W at 80℃ for 25min. After reaction, 1 mL of the reaction mixture was taken out for gel permeation chromatography (GPC) analysis. Then, the reaction mixture was cooled to room temperature (25℃) and adjusted to pH 7.0 with 10mol/L sodium hydroxide (NaOH) solution. Subsequently, the mixture was precipitated by adding anhydrous alcohol (500mL). The precipitations were collected by centrifugation for 15 min at 4000 rpm and lyophilized under 0.7Pa at minus 81℃ for 12h to yield 2.1g of powdered product and 70% yield. The product was referred to as WCOS. WCOS was prepared by our own laboratory according to above-mentioned conditions and equipment. 2.2.2 Degradation of chitosan under conventional heating The chitosan powder (3g) was introduced in an Erlenmeyer flask containing 2% acetic acid (100mL, that is 2mL glacial acetic acid plus 98mL distilled water). Then H2O2, whose final concentration is 3%, was added to the chitosan aqueous solution. The mixture solution was heated for degradation of chitosan by conventional heating. The degradation with conventional heating was carried out in a water bath at 80℃ with mechanical agitation for 180min. The experiments 3   

were employed under certain conditions. After reaction, 1 mL of the reaction mixture was taken out for gel permeation chromatography (GPC) analysis. The reaction mixture was cooled at room temperature (25℃) and adjusted to pH 7.0 with 10mol/L NaOH solution. Then the mixture was precipitated by anhydrous alcohol (500mL). The precipitations were collected by centrifugation for 15 min at 4000 rpm and lyophilized under 0.7Pa at minus 81℃ for 12h to yield 1.9g of powdered product and 63% yield. The product was referred to as ZCOS. WCOS, ZCOS was prepared according to the above-mentioned reaction and treatment conditions. 2.2.3 Characterization methods Average molar mass of degraded chitosans were measured by an Agilent 1260 gel permeation chromatography (Agilent Technologies, USA) equipped with a refractive index detector. Chromatography was performed on TSK G3000-PWXL columns, using 0.2 M CH3COOH/0.1 M CH3COONa aqueous solution as mobile phases at a flow rate of 1.0 mL/min with column temperature at 30 ◦C. The sample concentration was 0.4% (w/v). The standards used to calibrate the column were dextrans Mw 670,000, 270,000, 80,000, 50,000, 25,000, 12,000, 5000 and 1000 Da (Sigma, St. Louis, MO, USA). Determination method of degree of deacetylation as follows: Chitosan (0.3–0.5 g) was dissolved in 30 mL 0.1 M HCl at room temperature (20 ± 5 °C) in a 250 mL Erlenmeyer flask.

After  chitosan  dissolved  completely, then two drops of methyl orange indicator was added. 0.1 M NaOH was used to titrate the solution. At the final point of titration, the color changes from pink to orange yellow. The method was augmented to include the use of a pH meter to make the final titration point determination more precise. To calculate water content, 0.5 g chitosan was heated at 105 °C until a constant weight was reached. Three parallel samples were used. The percent of free NH 2 groups in chitosan was calculated as follows: NH 2 % = [(C 1 V 1 − C 2 V 2 ) × 0.016] / [G (100 − W)] ×100 Free NH 2 % = NH 2 %/9.94% × 100% Chitosan theoretic NH 2 content % = (16/161) × 100% = 9.94% C 1 : Concentration of HCl (M); C 2 : Concentration of NaOH (M); V 1 : the volume of HCl added (mL); V 2 : the volume of NaOH added by titration (mL); G: Sample weight (g); W: sample water content (%); 0.016: equal to NH 2 content (g) in 1 mL of 1 M HCl. The elemental analysis (C, H, and N) was performed on a Vario EL-III elemental analyzer. The percentages of carbon, hydrogen, and nitrogen were estimated. The mass spectrum (MS) analysis was performed on a LTQ ORBITRAP XL (Thermo Scientific). The samples were dissolved in CH3CN–H2O (1:1, v/v), and the solution was centrifuged. The supernatant was introduced into the mass spectrometer. The mass spectra were registered in the positive ion mode at a flow rate of 5µL min-1 .The capillary voltage was set to 3000 V, and capillary temperature was 275℃. All of the spectra were analyzed using Xcalibur. 2.2.4 Estimation of water-solubility The water solubility was assessed according to the method of Li et al. [2005]. The water solubility of chitooligosaccharides depending on various pH values was evaluated from the transmittance of the solution. Chitooligosaccharides (0.1 g) were dissolved in 1% HAc (100 mL), 4   

subsequently, the transmittance of the solution was recorded with the stepwise addition of 2mol/L NaOH (addition amount from 1mL to 50mL), on a Beijing Purkinje General TU-1810 ultraviolet spectrophotometer using a quartz cell with an optical path length of 1 cm at 600 nm. 2.2.5 Immunomodulatory effects of chitosan and their derivatives (COSs) in vitro 2.2.5.1 Macrophage phagocytosis capacity assay The purity of macrophages was tested by adherence [Li et al., 2008]. One-hundred microliter/well of macrophage suspension (RAW 264.7, 2-4×105 cells/well) and 20 µL/well of the different samples were plated in a 96-well culture plate and incubated at 37°C in a humidified atmosphere of 95% air/5% CO2. The experiments were divided into the blank control group without samples, only cells and culture medium and samples groups. The samples groups were chitosan groups, WCOS groups, ZCOS groups and YCOS groups. These samples were given at the concentrations of 0.1, 1, 10, and 100 µg/mL, respectively. Then, each well was pulsed with 5 mg/ml MTT (20 µL). After 4 h of incubation, the supernatant of the culture was carefully removed, 100 µl of DMSO was added to each well to terminate the reaction. Absorbance was determined at 492 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Tek, Winooski, VT, USA). 2.2.5.2 Splenic lymphocyte proliferation assay 2.2.5.2.1 Separation of lymphocyte The mouse was sacrificed by blood-letting eyeball with 75% alcohol immersion disinfection. Spleen was collected from sacrificed mice under aseptic conditions. Spleen tissue was ground into spleen cell liquid by homogenizer. The cell liquid successively passed through 180 mesh and 300 mesh fine steel sieve and the sieve was rinsed by RPMI-1640 medium. The homogeneous cell suspension was obtained and the erythrocytes were lysed with Tris- ammonium chloride lysis buffer. After centrifugation (380×g at 4 ℃ for 10 min), the pelleted cells were washed three times in phosphate-buffered saline (PBS) and resuspended in RPMI-1640 medium. Cell number was counted in haemocytometer by trypan blue dye exclusion solution and cell viability exceeded 95%. 2.2.5.2.2 Lymphocyte proliferation assay Splenic lymphocyte proliferation was assayed as described previously (Li, Jiao, et al., 2008). Cells were seeded at a density of 3-5×105 cells in a 96-well flat-bottom plate. In total, 3-5 × 105 cells were plated in 96-well plates with a 100 µl culture medium and then exposed to 20 µl different samples with or without mitogen (10 µg/mL ConA or 10 µg/mL LPS). The experiments were divided into the blank control group without sample and ConA and LPS. The samples groups (CTS, WCOS, ZCOS and YCOS, respectively) without ConA and LPS were given at a dose of 0.1, 1, 10, or 100 µg/mL. ConA or LPS group was given at a concentration of 10µg/mL. The sample groups (CTS, WCOS, ZCOS and YCOS, respectively) with ConA and LPS (10µg/mL) were given at a concentration of 0.1, 1, 10, or 100 µg/mL. The positive control group (Levamisole hydrochloride-levison) with 10 µg/mL ConA or 10 µg/mL LPS was given 10µg/mL. Cell viability was determined by MTT assay as mentioned above. 2.2.5.3 Assay of natural killer (NK) cells activity 5   

Splenic natural killer cell activity was evaluated by lactate dehydrogenase (LDH) release method [Konjevic, Jurisic, & Spuzic, 1997] with some modification. Briefly, YAC-1 cells, purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China), were rinsed 3 times by Hank’s solution and incubated with RPMI-1640 complete medium. Splenocytes prepared as described above were used as the effector cells. Ninety-microliter of splenocytes (5×106 cells/mL) and 90 µL of 2×106 cells/mL YAC-1 cells were mixed and seeded in 96-well round-bottom plates, then, 10 µl of samples solution was added to each well and incubated for 6 h. In the effector cells control group, YAC-1 cells were replaced by equivalent RPMI-1640 complete medium. Each experimental group was repeated in triplicate. Cell viability was determined by MTT assay as described above. 2.2.6 Immunomodulatory effects of chitosan and their derivatives in vivo Animals： Kunming rats were chosen to determine the immunomodulatory effects in vivo. Kunming rats, approximately the same age and with a body weight of 20–30 g, certificate number: SCXK-Liao 2010-0001, half male and half female, were purchased from Liaoning Chang sheng Biotechnology CO., LTD (XX city, Liaoning, China) and housed under controlled room of temperature (24 ± 5°C) and relative humidity (50 ± 10%) with 12 h light and 12 h darkness cycle. The rats were allowed free access to commercial stock diet and water. The experiments were divided into 12 groups, and each group consisted of 12 animals. All experimental procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Ethical Committee, and the protocols were approved by the Committee on the Ethics of Animal Experiments of the Institute of Oceanology, Chinese Academy of Sciences, Shandong, China. All efforts were made to minimize suffering, and the experimental animals were anesthetized using sodium pentobarbital before spleen and thymus were excised. The animals for the following experiments were pre-fasted overnight, but were allowed free access to water. 2.2.6.1 Effect of chitosan and their derivatives on humoral immunity The rats were divided into 12 groups and each group contained 12 animals. They are blank control group, Levamisole hydrochloride group (20 mg/Kg body weight/day), Chitosan group (100 mg/Kg body weight/day), WCOS group, YCOS group and ZCOS group, respectively. The concentration of WCOS group, YCOS group and ZCOS group was 33, 100 and 333 mg/Kg body weight/day, respectively.. All groups were taken by intragastric administration, once a day for 16 days. After all groups rats were treated by samples for 12 days, the animals were immunized by intraperitonially (i.p.) injecting 0.2ml of 2% SRBCs. Blood samples were collected in microlitre tubes from retroorbital vein puncture of individual animals of all the groups on day 4. Blood samples were centrifuged at 2000rpm for 10 min at 4 ℃ and serum was obtained. A microtechnique employing 96 -well microplates was used [Hudson and Hay, 1980]. Each well of the plate involved in 100 µl of serial two-fold dilutions of sera in saline and each well introduced an additional 100 µl of 0.5% (v/v) SRBC suspension in saline, then mixed well. After incubating the mixtures for 3 h at 37 ℃ the hemagglutinating capacity was calculated: ∑=S1+2S2+3S3+...+nSn, where S was defined as the degree of agglutinating, n was defined as dilute rate.

6   

2.2.6.2 Assessment of chitosan and their derivatives on delayed type hypersensitivity (DTH). Assessment of chitosan and their derivatives on cell mediated immune functions was performed by delayed type hypersensitivity measured by Mantoux test. The treatment was mentioned in Section 2.2.1. On the 5th day of immunization, the animals were again challenged with 20µl of a challenging dose of 1×108 cells SRBCs (20%, v/v) in the right hind paw [Mangathayarua et al., 2009]. The thickness of the paw was measured with sphaeromicrometer (pitch 0.01mm) at 0, 24h of SRBCs challenge. DTH response was expressed as the difference of between pre and post-challenge paw thickness. 2.2.6.3 Effect of chitosan and their derivatives on mononuclear phagocytic system function The treatment was mentioned in Section 2.2.6.1. 2.2.6.3.1 Spleen and thymus indices On the sixteenth day, the food was deprived. Twenty-four hours later, the rats were sacrificed. The spleen and thymus were excised from each animal. The body, spleen and thymus were immediately weighted. The spleen and thymus indices were calculated according to the following formula: spleen or thymus indices (mg/g)=spleen or hymus weight (mg)/body weight (g). 2.2.6.3.2 Mononuclear phagocytic system (MPS) function assay The function of the MPS was analyzed using a carbon clearance test [Chen et al., 2010]. Thirty min after the last dose of the samples, Indian ink (0.05mL/10g body weight) was injected into the lateral tail vein of mice. After Indian ink injection, 20 µL of blood was taken from the retro-orbital venous plexus of each mouse at 1 and 5 min intervals. The blood was mixed with 2 mL 0.1% (m/v) Na2CO3 in a tube. The mixture was measured at the optical density of 680 nm. The rate of clearance (K value) K= (lgA1-lgA2)/(t2-t1) Where A1 is the optical density at t1 and A2 is the optical density at t2. Phagocytic index α=(

)/(B+C)

Where A is the body weight, B is spleen weight, C is thymus weight. 2.2.7 Statistical Analysis All of the data were expressed as mean ± standard deviation (SD) of three replicates and were analyzed statistically by one-way analysis of variance using SPSS version 10.0 software. The statistical significance between the means of the experimental and control studies was established by Student’s t-test. The results were considered to be significant if p < 0.05, p < 0.01 or p < 0.001. Results: 3.1 Properties and Elemental analysis of chitosan and three degradation resultants As shown in Table 1, average molar mass of chitosan derivatives by different degradation methods was almost equivalent, especially WCOS and ZCOS. From Mw/Mn, we found dispersion degree of WCOS degraded by microwave irradiation was the best. Molecular weight played a critical role for their activities. Therefore, in this experiment, the influence of molecular weight was almost negligible, especially WCOS and ZCOS. Elemental analysis results of parent and degraded chitosan are also listed in Table 1. As 7   

shown in Table 1, the H/C mass ratio of ZCOS was almost the same as chitosan, however, the percent of N of WCOS was almost the same as chitosan. Previous studies reported that deamination side reaction was occurred during oxidative degradation of chitosan [Qin et al, 2002; Duy et al, 2011]. Our results confirmed the change of nitrogen in the degradation reaction. However, the reason for the inconsistency of YCOS degraded by enzyme and chitosan maybe the effect of the presence of inactivated enzyme. However, further investigation is needed to determine the specific reason. 3.2 Solubility of chitosan and degraded chitosan Compared with the parent chitosan, three kinds of chitooligosaccharides YCOS, WCOS and ZCOS showed higher water solubility over a wide pH range. Chitosan with high molecular weights was only dissolved in acid solution and separated out over pH 6. Water insolubility limited its wide application. However, chitooligosaccharides have the better solubility among pH 1-14 (data not shown). This maybe reduces the intermolecular interactions and crystallinity [Li et al, 2005]. The water solubility of chitooligosaccharides would lead to low viscosity and high concentration of their solution, which was favorable to the wider application of chitosan and reduced the environment pollution, which was caused by acid, alkali and organic solvents. 3.3 ESI/MS analysis of degraded chitosan ESI/MS is a powerful method for the molecular weight and distribution of polymerization degree analysis of oligosaccharides. It can give the exact molecular weight of the analyte and be extensively applied to determine the distribution of polymerization degree of oligosaccharides [Volpi, 2007; Galeotti and Volpi, 2011; Yang et al., 2010]. The peaks were [M + H]+ ion-peaks (e.g. 3-mers: calculated mass, 179 (C6H13O5N) + 2 × 161 (C6H11O4N) + 1 (H, the adduct ion) = 502 mass units, experimental mass, 502.23 mass units), or [M + one acetyl group]+ (e.g. 4-mers: calculated mass, 179 (C6H13O5N) + 3 × 161 (C6H11O4N) + 1 (H, the adduct ion) +43 (C2H3O) = 705 mass units, experimental mass, 705.31 mass units) or [M + two acetyl group]+ (e.g. 3-mers: calculated mass, 179 (C6H13O5N) + 2 × 161 (C6H11O4N) + 1 (H, the adduct ion) +2 ×43 (C2H3O) = 587 mass units, experimental mass, 586.25 mass units). The difference between the mass/charge ratios (m/z) of adjacent peaks was 161 Da, which was exactly the molecular mass of a GlcN residue. As shown in Fig. 2, the chitooligosaccharides obtained by different preparation methods

were  analyzed  by  ESI‐MS  with  Fig.  1A  showing  the  ESI‐MS  spectrum  of  ZCOS. It was clearly observed that nine peaks corresponding to the glucosamine oligomers and acetylglucosamine oligomers with one acetyl group or a little bit of two acetyl groups with DP 1–5 were monitored in the spectrum. The mass/charges (m/z) of the main peaks were 180.09, 341.16, 502.23, 663.30, and 412.69 (z=2), respectively, these peaks were [M+H+] ion-peaks with 161 Da mass larger than the peak ahead, which was exactly the molecular mass of a GlcN residue. The mass/charges (m/z) of the main peaks were 383.17, 544.24, 705.31 and 866.36, respectively, these peaks were [M + one acetyl group]+ ion-peaks and the main peak 586.25 was [M + two acetyl group]+ ion-peak. Fig. 1B showed the ESI-MS spectrum of WCOS. It mainly was made up of two sugar to six sugar with or without acetyl group. The m/z values of 341.16, 502.23, 332.15 (z=2), 663.30, 412.69 (z=2) could be assigned to the [M + H]+ ions of glucosamine dimers, trimers, tetramers and pentamers, respectively. The peaks of 383.17 (Z=2), 544.24 and 866.37 were [M + one acetyl group] ions of dimers, trimers and pentamer with one acetyl group. The peaks of 8   

586.25, 747.32 were [M + two acetyl group] ions of trimers and tetramers with two acetyl group. Moreover, WCOS has hexamers, which the peak 1008.41 was assigned to the [M + Na]+ ion, while monomers were not detected. For YCOS, as  shown  in  Figure  1C, it mainly contained glucosamine trimers, tetramers and pentamers. The m/z values of 502.23, 251.62 (z=2), 663.30, 332.15 (z=2), 824.37, 412.69 (z=2) could be assigned to the [M + H]+ ions. Monomers were not also detected for YCOS. 3.5 Immunomodulatory effects of chitosan and their derivatives in vitro 3.5.1 Effect of chitosan and their derivatives on macrophage phagocytosis

As shown in Table 2, each sample had differently enhancing effects on macrophage phagocytosis at 0.1-100 µg/ml. Compared with the normal control group, the activity of CTS on macrophage phagocytosis had significant increase (p<0.05, p<0.05) at the concentration of 1 or 10 µg/mL. Treatment with YCOS or ZCOS at the concentration of 10 µg/ml and WCOS at the concentration of 1 µg/ml caused significant increase (p<0.001, p<0.05, p<0.01) on macrophage phagocytosis. Therefore, these results indicated that chitosan and their derivatives can mediate macrophage phagocytosis at the certain concentration. 3.5.2 Effect of chitosan and their derivatives on splenic lymphocyte proliferation The effects of chitosan and their derivatives on normal (without mitogen) and mitogen-induced splenic lymphocyte proliferation were investigated at the concentration ranging from 0.1 to 100µg/ml, as shown in Table 3. Table 3 showed that CTS and YCOS without mitogen could increase splenocyte lymphocyte proliferation compared to the control. Treatment with CTS at 0.1, 1,and 10 µg/ml caused a significant splenocyte lymphocyte proliferation (p<0.001, p<0.001, p<0.05). Moreover, YCOS significantly promoted the proliferation of splenocytes at the concentration of 1 µg/ml and 10 µg/ml (p<0.01, p<0.05). However, WCOS and ZCOS do not have a significant effect on splenocyte lymphocyte proliferation compared to the control. As shown in Tables 3, both ConA and LPS could significantly stimulate lymphocyte proliferation compared with the normal control (p<0.001, p<0.001). The effect of all samples on the proliferation response of lymph-node lymphocytes with the T-cell mitogen ConA was investigated. Compared with the ConA group, the levison could significantly increase the splenocyte lymphocyte proliferation. Treatment with CTS at the concentrationof 0.1µg/ml or 100µg/ml of ConA caused a significant lymphocyte proliferation (p<0.05, p<0.001). However, other treatments do not have a significant effect on proliferation of splenocytes. Almost similar results were obtained when the effect of chitosan and their derivatives on the proliferation response of spleen lymphocytes to the B-cell mitogen LPS was assayed. Compared with the LPS group, the positive group levison also could significantly increase the lymphocyte proliferation. Effects of WCOS and ZCOS with LPS on lymphocyte proliferation had slight enhancement at all concentrations of groups. CTS (100 µg/ml) and YCOS (100 µg/ml) significantly increased cell proliferation compared with the LPS control (p<0.001, p<0.01). However, other concentration groups of YCOS (0.1, 1, 10 µg/ml) displayed slight depressant effects. 9   

3.5.3 Effect of chitosan and their derivatives on NK cell cytotoxicity NK cells are a type of lymphocytes and are part of the first line of innate defense against cancer cells and virus-infected cells [Moretta et al, 2001]. The effects of chitosan and their derivatives on NK cell were investigated with the concentration ranging from 0.1 to 100µg/ml, as shown in Table 4. Except 0.1µg/ml groups of YCOS and WCOS, other samples including levison significantly enhanced NK cell activation compared with the normal control group (p<0.001). More, obviously increasing differences among ZCOS, WCOS, YCOS and CTS were also observed, which indicated that ZCOS was the most efficient for inducing NK cell activation, and WCOS was the second. 3.6. Assessment of chitosan and their derivatives on humoral immune functions in vivo 3.6.1 Effect of chitosan and their derivatives on circulating hemaggultinating antibody titer (HA titer)

In this paper, the hemaggultinating antibody titer (HA titer) assay was used to assess the effect of chitosan and their derivatives on humoral immune response. As shown in Table 5, all groups could increase the HA titer compared with the control group. Treatment with CTS (100 mg/kg), WCOS (100 mg/kg), YCOS (33 mg/kg) and ZCOS (33 mg/kg) showed a significant rise in HA titer values at 16th day after immunization. 3.6.2 Assessment of chitosan and their derivatives on delayed type hypersensitivity (DTH)

The DTH reaction is a cell-mediated pathologic response involved with T cell activation and the production of many cytokines [Kobayashi et al., 2001]. The effect of chitosan and their derivatives on T-cell mediated DTH reaction is shown in Table 6. The results showed that only WCOS at a dose of 333 mg/kg can significantly increase DTH reaction compared with control and the positive control. Moreover, the effect of WCOS on DTH reaction increased at a dose dependent manner. 3.6.3 Effects of chitosan and their derivatives on spleen and thymus indices and phagocytotic function of the mononuclear phagocyte system

The morphological changes in the spleen and thymus were examined to evaluate the effects of chitosan and their derivatives on the immune organs. As shown in Table 7, the WCOS-treated group showed a significant increase (p<0.05) in the spleen indices at a concentration of 100mg/kg and a slight increase in the thymus index compared with the normal control group. However, other treatment groups can slightly increase the spleen and thymus indices, but the effect is not statistically significant. At the same time, to evaluate the effect of chitosan and their derivatives on the mononuclear phagocytes system, a carbon clearance test was performed. The carbon particles in India ink were 10   

cleared by macrophage when India ink was introduced directly into the mononuclear phagocyte system. Table 7 showed that the phagocytic indices α in mice. Compared with the normal control group, the phagocytic indices α in the groups treated with YCOS (333 mg/kg), WCOS (100 mg/kg and 333 mg/kg) and ZCOS (100 mg/kg) were significantly elevated (p<0.01, p<0.05, p<0.05, p<0.001). The positive group can also significantly increase the phagocytic indices α (p<0.05), however, YCOS, WCOS and ZCOS were more efficient than the positive group. Discussion: Our previous studies and many other studies have reported that chitooligosaccharides degraded from chitosan are relatively nontoxic and do not cause significant side effects. These side effects are a major problem of chemically synthesised compounds. In China, chitooligosaccharides were approved as a new food resource in 2014 by the National Health and Family Planning Commission [2014]. Therefore, chitooligosaccharides can be used as an additive in the food industry. Chitooligosaccharides are ideal candidates for therapeutics involving immunomodulatory activity. The immunomodulatory effects of chitooligosaccharides have been extensively reported and health products containing chitooligosaccharides have been recently developed. However, the immunomodulatory effects of the monomer composition of chitooligosaccharides obtained by oxidative degradation, microwave irradiation, or enzymatic hydrolysis have not been determined. As shown by electrospray ionisation mass spectrometry (Figure 1), ZCOS obtained by oxidative degradation was composed of monosaccharides to pentasaccharides, with mainly disaccharides and trisaccharides with or without acetylaion. WCOS obtained by microwave irradiation was composed of disaccharides to hexasaccharides, with mainly disaccharides, trisaccharides, tetrasaccharides, and pentasaccharides. Almost all of the saccharides were monomers without an acetyl group, monomers with an acetyl group, and monomers with two acetyl groups. Furthermore, WCOS contained small amounts of hexasaccharides that were reported to possess obvious activity. YCOS was mainly composed of trisaccharides to pentasaccharides without acetyl groups. Many studies have reported that monomer composition and acetyl groups are two important factors for chitooligosaccharide activity. In our study, the effects of chitooligosaccharides obtained by different degradation methods were determined on macrophages, B cells, T cells, and natural killer (NK) cells in vitro, and on cellular and humoral immunity in vivo. Macrophages are the first line of defence in the immune response to foreign invaders, and also present antigen-derived peptide fragments to T lymphocytes, resulting in activation of immunologically competent T cells [Al-Daccak, Mooney, and Charron, 2004]. Activated macrophages are therefore considered to be important immunoctyes against tumour growth [Cao et al., 2010]. The results of the present study showed that chitosan and its degradation products (YCOS, WCOS, and ZCOS) obtained using different reaction methods could induce macrophage activity at specific concentrations. Moreover, WCOS could stimulate the macrophage activity at a low concentration (1 μg/mL; P < 0.01). Splenic lymphocyte proliferation induced by concanavalin A (ConA) in vitro has been used to evaluate T lymphocyte activity, and splenic lymphocyte proliferation induced by lipopolysaccharide (LPS) has been used to evaluate B lymphocyte activity [Sun et al., 2009]. T cells are involved in cell-mediated immunity, and B cells are primarily responsible for humoral immunity. The present results showed that high-molecular-weight chitosan could significantly 11   

stimulate spleen lymphocyte proliferation, and chitosan could enhance the proliferation of T cells treated with ConA at a concentration of 0.1 μg/mL and 100 μg/mL, and significantly increase the proliferation of B cells treated with LPS at a concentration of 100 μg/mL. These results showed that in vitro, chitosan can increase humoral immunity and cell-mediated immunity. However, low molecular weight degraded products only slightly enhanced splenic lymphocyte proliferation, and the products at certain concentrations even had a negative effect on mitogen-induced proliferation. The molecular weight is therefore an important parameter involved in in vitro lymphocyte proliferation activity. The high molecular weight forms of chitosan exhibited the best splenic lymphocyte proliferation activity in vitro. However, we found that in vivo immunomodulation activities of chitosan were lower than that of degraded chitosan. In this study, an NK cell assay was used to evaluate the immunomodulatory activities of chitosan and its degradation products in vitro. The function of NK cells is similar to cytotoxic T lymphocytes (CTLs). Unlike CTLs, killing by NK cells is nonspecific; they can destroy target cells without the help of major histocompatibility complex (MHC)-dependent recognition or prior sensitization, and they do not need to recognize the antigen/MHC on the target cell. The NK cell activity assay is a routine in vitro method for analysis of a patient’s cellular immune response, and can also be used to test the antitumor activities of possible drugs [Zhang et al., 2005]. In this study, chitosan and three chitooligosaccharides were found to significantly enhance the killing activity of NK cells at 0.1–100 μg/mL. Moreover, the optimal concentration was 1 μg/mL for three chitooligosaccharides, and showed that a high concentration is not essential for strong stimulating effects. At low concentrations, chitosan and chitooligosaccharides enhance nonspecific cytolytic activities and may modulate NK cells to inhibit infection and tumour initiation. The haemagglutinating antibody titre test was used to evaluate the effect of chitosan and its products degraded by different methods on the humoral immune response. Antibody molecules, consisting of B lymphocytes and plasma cells, are central to humoral immune responses. In this study, compared with the control group and the positive group, CTS (100 mg/kg), WCOS (100 mg/kg), YCOS (33 mg/kg), and ZCOS (33 mg/kg) significantly increased the haemagglutinating antibody titre. The results indicated that chitosan and its degradation products can induce immunostimulation through humoral immunity. The delayed-type hypersensitivity reaction directly correlates with cell-mediated immunity involving T lymphocytes and lymphokines. There is evidence to suggest that the delayed-type hypersensitivity response is important in host defence against parasites and bacteria that can live and proliferate intracellularly. In this study, only WCOS produced by microwave irradiation degradation increased delayed-type hypersensitivity. This increase showed that WCOS had a stimulatory effect on T lymphocytes and lymphokines. The spleen and thymus are important organs involved in immune functions. The spleen represents a major secondary lymphoid organ involved in elicitation of immune responses. In this study, WCOS significantly increased the spleen index. Phagocytes participate in the innate immune response, and phagocytosis represents an important immune defence mechanism. The carbon clearance test was used to evaluate this system. The reticuloendothelial system is a diffuse system composed of phagocytic cells. When colloidal carbon particles in the form of ink are injected directly into the systemic circulation, the rate of clearance of carbon from the body by macrophages can be characterized by an exponential equation [Gokhale et al., 2003]. In this study, CTS and their derivatives, especially WCOS, all 12   

significantly increased the phagocytic index. According to the previously mentioned results, we found that chitosan and its degraded products, especially WCOS, significantly stimulated the immune system by acting through cellular and humoral immunities. Although chitosan has immune stimulatory activity, it only acted in vitro. It is possible that its high molecular weight could not be metabolized and absorbed in vivo. Among the three degradation products (ZCOS, WCOS, and YCOS), the immunomodulatory effect of WCOS was the strongest. Compared with YCOS and ZCOS, it significantly increased the spleen index and significantly stimulated the delayed-type hypersensitivity reaction. As shown by the mass spectrometry results, WCOS was composed of 2–6 sugar monomers, with or without acetyl groups. These monomers all contained one or two acetyl groups. However, compared with WCOS, ZCOS contained 1–5 sugar monomers and lacked hexamers or monomers with 2 acetyl groups, and YCOS consisted of 3–5 sugar monomers and lacked hexamers or monomers with 1 or 2 acetyl groups. Many studies have reported that compared with other monomers, chitohexaose and hexa-N-acetylchitohexaose possess stronger biological activities [Suzuki et al., 1986; Qin et al., 2002; Xiong et al., 2009]. Our results showed that the partially acetylated chitotrioses had greater antioxidant activities than fully acetylated and deacetylated chitotrioses [Li et al., 2013]. The better immunomodulatory effects of WCOS might therefore result from the hexamer and acetyl groups in the molecule. We separated seven highly purified (2–8) glucosamine oligomers with single degree of polymerization. Moreover, our purification procedure produced over 300 mg each of dimers, trimers, and tetramers, and 100 mg each of pentamers, hexamers, heptamers, and octamers in sufficient quantities for determination of biological activities [Li et al., 2013]. Therefore, for follow-up work, we would like to use pure oligosaccharide monomer to do the immune activity and verify which individual sugar has the strongest effect, to support the findings of this study. In this paper, an interesting point was the immunomodulatory effects of all determination samples did not appear to be a dose dependent relationship. This result was in accord with Mohammad Yousef et al.’s result [2012]. Mohammad Yousef et al.’s result showed that grading of the magnitude of protection by COS was 10 mg/kg/day = 20 mg/kg/day > 5 mg/kg/day > 50 mg/kg/day >1 mg/kg/day > 100 mg/kg/day. They called this the bell-shaped pattern of dose-response relationship of COS. They thought that this biphasic effect of COS could be attributed to the capacity of COS to stimulate two different signaling pathways resulting in opposing effects, so called functional antagonism. The sum of the two dose-response curves therefore determined the net effect of COS and yielded the bell-shaped dose-response curve. Based on our results, chitooligosaccharides produced by microwave irradiation had the best immunomodulation activity, indicating that it was the best method to prepare chitooligosaccharides. Future studies will involve in identification of the monomers possessing the best activity, and identification of the optimal degree of acetylation. These studies will further characterize the roles of each monomer and acetyl group to determine protocols for the production of the most active chitooligosaccharides.

Conflicts of Interest The authors declare that they have no conflict of interest. 13   

Acknowledgement We gratefully acknowledge Shenyang Pharmaceutical University for the activities of products (Liaoning, China). This study was supported by Shandong Provincial Science and Technology Major Project, China (Grant No.2015ZDZX05003), the Science and Technology Development Program of Shandong Province (No. 2014GGH215006), and the commonweal item of State Oceanic Administration People’s Republic of China (201405038-2), and the Qingdao science and technology plan (No.14-2-3-47-nsh ), The Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology（No.2015ASKJ02）. Conference Aam, B.B., Heggset, E.B., Norberg, A.L., Sørlie, M., Vårum, K.M., Eijsink, V.G.H. (2010) Production of Chitooligosaccharides and Their Potential Applications in Medicine. Marine Drugs, 8, 1482–1517. Al-Daccak, R., Mooney, N., Charron, D. (2004) MHC class II signaling in antigen-presenting cells. Current Opinion in Immunology, 16(1), 108-113. Cao, W., Li, X. Q., Wang, X., Li, T., Chen, X., Liu, S. B., Mei, Q. B. (2010) Characterizations and anti-tumor activities of three acidic polysaccharides from Angelica sinensis (Oliv.) Diels. International Journal of Biological Macromolecules, 46, 115-122. Chang, K.L.B., Tai, M.C., Cheng, F.H. (2001) Kinetics and Products of the Degradation of Chitosan by Hydrogen Peroxide. Journal of Agricultural and Food Chemistry, 49, 4845–4851. Chen, J. R., Yang, Z. Q., Hu, T. J., Yan, Z. T., Niu, T. X., Wang, L. (2010) Immunomodulatory activity in vitro and in vivo of polysaccharide from Potentilla anserine. Fitoterapia, 81(8), 1117-1124. Duy, N.N., Phu, D.V., Anh, N.T., Hien, N.Q. (2011) Synergistic degradation to prepare oligochitosan by γ-irradiation of chitosan solution in the presence of hydrogen peroxide. Radiation Physics and Chemistry, 80, 848–853. Feng, J., Zhao, L. H., Yu, Q. Q. (2004) Receptor-mediated stimulatory effect of oligochitosan in macrophages. Biochemical and Biophysical Research Communications, 317, 414-420. Galeotti, F., Volpi, N. (2011) Online Reverse Phase-High-Performance Liquid Chromatography-Fluorescence Detection-Electrospray Ionization-Mass Spectrometry Separation and Characterization of Heparan Sulfate, Heparin, and Low-Molecular Weight-Heparin Disaccharides Derivatized with 2-Aminoacridone. Analytical Chemistry, 83, 6770–6777. Gokhale, A. B., Damre, A. S., Saraf, M. N. (2003) Investigations into the immunomodulatory activity of Argyreia speciose. Journal of Ethnopharmacology, 84, 109-114. Hudson, L., Hay, P. (1980) Chapter 5: Antibody interaction with antigen. In: Thomas, A. (Ed.), Practical Immunology. Blackwell, Edinburgh, P. 131. Heggset, E.B., Dybvik, A.I., Hoell, I.A., Norberg, A.L., Sorlie, M., Eijsink, V.G.H., Varum, K.M. (2010) Degradation of Chitosans with a Family 46 Chitosanase from Streptomyces coelicolor A3(2). Biomacromolecules, 11, 2487–2497. Huang, M., Khor, E., Lim, L.Y. (2004) Uptake and Cytotoxicity of Chitosan Molecules and Nanoparticles: Effects of Molecular Weight and Degree of Deacetylation. Pharmaceutical Research, 21, 344–353. 14   

Ismail, S., Asad, M. (2009) Immunomodulatory activity of Acacia catechu syed ismail and mohammed asad. Indian Journal of Physiology and Pharmacology, 53 (1), 25-33. Khor, E., Lim, L.Y. (2003) Implantable applications of chitin and chitosan. Biomaterials, 24, 2339–2349. Kobayashi, K., Kaneda, K., Kasama, T. (2001) Immunopathogenesis of delayed-type hypersensitivity. Microscopy Research and Technique, 53, 241-245. Konjevic, G., Jurisic, V., Spuzic, I. (1997) Corrections to the original lactate dehydrogenase (LDH) release assay for the evaluation of NK cell cytotoxicity. Journal of Immunological Methods, 200 (1-2), 199-201. Li, J., Du, Y., Yang, J., Feng, T., Li, A., Chen, P. (2005) Preparation and characterisation of low molecular weight chitosan and chito-oligomers by a commercial enzyme. Polymer Degradation and Stability, 87, 441–448. Li, X., Jiao, L.L., Zhang, X., Tian, W. M., Chen, S., Zhang, L. P. (2008) Antitumor and immunomodulating activities of proteoglycans from mycelium of Phellinus nigricans and culture medium. International Immunopharmacology, 8, 909-915. Li, K.C., Liu, S., Xing, R.E., Qin, Y.K., Li, P.C. (2013) Preparation, separation and antioxidant activity of partially N-acetylated chitotriose. Carbohydrate Polymers, 92, 1730-1736. Li, K.C., Liu, S., Xing, R.E., Yu, H.H., Qin, Y.K., Li, R.F., Li, P.C. (2013) High-resolution separation of homogeneous chitooligomers series from 2-mers to 7-mers by ion-exchange chromatography. Journal of Separation Science, 36, 1275-1282. Lim, T. S., Na, K., Choi, E. M., Chung, J. Y., Hwang, J. K. (2004) Jmmunomodulating activities of polysaccharides isolated from Panax ginseng. Journal of Medicinal Food, 7, 1-6. Mangathayarua, K., Vmadevi, M., Reddy, V. (2009) Evaluation of the immunomodulatory and DNA protective activities of the shoots of Gynodon dactylon. Journal of Ethnopharmacology, 123, 181-184. Mohammad, Y., Rath, P., Sunhapas, S., Varanuj, C., Chatchai, M. (2012) Chitosan oligosaccharide as potential therapy of inflammatory bowel disease: Therapeutic efficacy and possible mechanism of action. Pharmacological Research, 66, 66-79. Moretta, L., Bottino, C., Cantoni, C., Mingari, M. C., Moretta, A. (2001) Human natural killer cell function and receptors. Current Opinion in Pharmacology , 1, 387-391. Muzzarelli, R.A.A. (2010) Chitins and Chitosans as Immunoadjuvants and Non-Allergenic Drug Carriers. Marine Drugs, 8, 292–312. National Health and Family Planning Commission. Announcement (2014 sixth) on the approval of 6 new food ingredients such as chitooligosaccharide. http://www.nhfpc.gov.cn/sps/s7890/201405/367ce408981e4807809e107417b3d361.shtml. No, H.K., Park, N.Y., Lee, S.H., Meyers, S.P. (2002) Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. International Journal of Food Microbiology, 74, 65–72. Qin, C.Q., Du, Y.M., Xiao, L. (2002) Effect of hydrogen peroxide treatment on the molecular weight and structure of chitosan. Polymer Degradation and Stability, 76, 211–218. Qin, C., Du, Y., Xiao, L., Li, Z., Gao, X. (2002) Enzymic preparation of water-soluble chitosan and their antitumor activity. International Journal of Biological Macromolecules, 31, 111–117. Sun, Y., Liang, H., Zhang, X., Tong, H., Liu, J. (2009) Structural elucidation and immunological activity of polysaccharide from the fruiting body of Armillaria mellea. Bioresource Technology, 15   

100, 1860-1863. Suzuki, K., Mikami, T., Okawa, Y., Tokoro, A., Suzuki, S., Suzuki, M. (1986) Antitumor effect of hexa-N-acetylchitohexaose and chitohexaose. Carbohydrate Research, 151, 403-408. Trombotto, S., Ladaviere, C., Delolme, F., Domard, A. (2008) Chemical Preparation and Structural Characterization of a Homogeneous Series of Chitin/Chitosan Oligomers. Biomacromolecules, 9, 1731–1738. Volpi, N. (2007) On-Line HPLC/ESI-MS Separation and Characterization of Hyaluronan Oligosaccharides from 2-mers to 40-mers. Analytical Chemistry, 79, 6390–6397. Xia, W., Liu, P., Zhang, J., Chen, J. (2011) Biological activities of chitosan and chitooligosaccharides. Food Hydrocolloids, 25, 170–179. Xing, R.E., Liu, S., Guo, Z.Y., Yu, H.H., Wang, P.B., Li, C.P., Li, Z., Li, P.C. (2005) Relevance of molecular weight of chitosan and its derivatives and their antioxidant activities in vitro. Bioorganic and Medicinal Chemistry, 13, 1573–1577. Xing, R.E., Liu, S., Yu, H.H., Guo, Z.Y., Wang, P.B., Li, C.P., Li, Z., Li, P.C. (2005) Salt-assisted Acid Hydrolysis of Chitosan to Oligomers under Microwave Irradiation. Carbohydrate Research， 340，2150－2153. Xiong, C., Wu, H., Wei, P,, Pan, M., Tuo, Y., Kusakabe, I., Du, Y. (2009) Potent angiogenic inhibition effects of deacetylated chitohexaose separated from chitooligosaccharides and its mechanism of action in vitro. Carbohydrated Research, 344, 1975-1983. Yang, Z.M., Yi, Y.T., Gao, C.C., Hou, D.K., Hu, J., Zhao, M.Y. (2010) isolation of inulin-type oligosaccharides from Chinese traditional medicine: Mor.inda officinalis How and their characterization using ESI-MS/MS. Journal of Separation Science, 33(1), 120–125. Zhang, J., Sun, R., Wei, H., Tian, Z. (2005) Antitumor effects of recombinant human prolactin in human adenocarcinoma-bearing SCID mice with human NK cell xenograft. International Immunopharmacology, 5, 417-425. Zhu, X. L., Chen, A. F., Lin Z. B. (2007) Ganoderma lucidum polysaccharides enhance the function of immunological effector cells in immunosuppressed mice. Journal of Ethnopharmacology, 111, 219-226.

16   

17   

 

Figure 1 ESI-MS spectrum of chitooligosaccharides. The peaks were [M+H+] ion-peaks of glucosamine oligomers with 161 Da mass or ion-peaks with an acetyl group or two acetyl groups. A: ZCOS, B: WCOS, C:YCOS.

Table 1 Properties and Elemental analysis result of original chitosan (CS) and degraded chitosans (YCOS, WCOS and ZCOS)   samples 

Mw  ×10−3  Da 

Mw/Mn 

N% 

C% 

H% 

N/C 

H/C 

CS 

658 

 

6.76 

43.05 

7.11 

0.157 

0.165 

YCOS 

1.11 

3.7454 

6.27±0.05  41.02±0.07  6.00±0.40  0.153±0.002  0.146±0.010

WCOS 

1.46 

2.1994 

6.72±0.03  40.09±0.03  6.08±0.15  0.168±0.001  0.152±0.004 

ZCOS 

1.36 

2.2121 

6.44±0.13  39.26±0.10  6.36±0.50  0.164±0.003  0.162±0.013

    Table 2 Effect of chitosan and their derivatives on macrophage phagocytosis (Mean±SE) Concentration (μg/mL)

CTS

YCOS

WCOS

ZCOS

0

100.00±2.83

100.00±2.83

100.00±2.83

100.00±2.83

0.1

107.55±4.11

117.69±2.70

112.24±8.85

117.46±7.73

1

118.49±2.02*

103.31±2.29

125.68±0.52**

115.19±5.89

10

120.11±4.74*

137.07±1.08***

111.67±3.84

117.24±1.40*

100

108.99±4.00

106.11±7.70

110.09±4.46

108.84±4.99

*p < 0.05, ** p < 0.01, *** p < 0.001 compared with control group.     Table 3 The effects of chitosan and their derivatives on normal (without mitogen) and mitogen-induced (ConA or LPS) splenic lymphocyte proliferation (Mean±SE) Group

Concentratio

CTS

YCOS

WCOS

ZCOS

n (μg/mL) Without

0

100.00±0.90

100.00±0.90

100.00±0.90

100.00±0.90

mitogen

0.1

109.60±1.59***

101.94±1.18

102.42±1.18

103.30 ±0.58

1

107.47±0.83***

106.01±2.13**

101.26±0.29

99.81±1.51

10

105.24±1.65*

105.82±2.09*

100.68±1.10

100.87±0.68

100

104.66 ±1.27

100.78±1.56

103.30±1.05

103.49±0.54

18   

Con

0

86.43±2.18

86.43±2.18

86.43±2.18

86.43±2.18

ConA

10

100.00±1.46###

100.00±1.46###

100.00±1.46###

100.00±1.46###

Levison

10

108.09±1.25**

108.09±1.25**

108.09±1.25**

108.09±1.25**

ConA+Sampl

0.1

107.10±2.98*

103.89±1.25

98.47±2.13

101.68±1.82

e

1

105.44±0.88

103.12±1.33

99.03±0.40

99.80±0.19

10

104.89±3.92

97.70±0.48

101.24±0.48

97.70±0.96

100

110.96±1.05***

103.78±1.89

105.66±0.77

106.32±1.25

Con

0

65.01±0.58

65.01±0.58

65.01±0.58

65.01±0.58

LPS

10

100.00±1.20###

100.00±1.20##

100.00±1.20##

100.00±1.20##

#

#

#

Levison

10

106.84±1.19*

106.84±1.19*

106.84±1.19*

106.84±1.19*

LPS+Sample

0.1

101.20±0.62

99.46±0.74

101.01±1.36

101.66±1.42

1

101.92±0.36

99.78±0.80

101.40±1.76

102.43±1.62

10

104.31±0.22

99.33±1.89

101.92±1.04

103.02±1.41

100

109.88±1.64** *

106.78±2.83* *

101.07±3.29

100.43±1.59

Without mitogen groups: *p < 0.05, ** p < 0.01, *** p < 0.001 compared with control group. ConA groups: ### p < 0.001, compared with control group. And * p < 0.05, ** p < 0.01, *** p < 0.001, compared with ConA group. LPS groups: ### p < 0.001, compared with control group. And * p < 0.05, ** p < 0.01, *** p < 0.001, compared with LPS group. Table 4 Effect of chitosan and their derivatives on NK cell activation (Mean±SE)

Group

Concentration

CTS

YCOS

WCOS

ZCOS

31.45±2.71

31.45±2.71

(μg/mL) Con

0

31.45±2.71

31.45±2.71

Levison

10

72.07±1.80***

72.07±1.80***

72.07±1.80***

72.07±1.80***

Sample

0.1

57.05±6.17**

44.32±0.66

47.88±4.67

72.12±1.86***

1

67.95±3.02***

68.94±8.91***

84.39±7.48***

90.45±3.88***

10

57.88±4.86**

56.14±0.13**

63.48±5.72***

74.69±9.06***

100

76.67±3.95***

76.52±3.47***

84.69±4.61***

88.18±5.36***

** p < 0.01, *** p < 0.001, compared with control group.

19   

Table 5 Effect of chitosan and their derivatives on circulating hemaggultinating antibody titer (Mean±SE) Group

Dose (mg/kg)

Number (n)

Antibody titer (16d)

Con

0

11

36.00±10.28

Levison

20

11

55.91±12.70

CTS

100

11

70.00±9.07*

WCOS

33

12

46.17±13.71

100

12

73.17±15.69*

333

12

62.83±13.00

33

12

73.92±13.07*

100

12

67.25±15.50

333

10

58.70±12.35

33

10

74.50±10.76*

100

12

46.25±12.94

333

12

66.67±17.96

YCOS

ZCOS

* p < 0.05, compared with control group.     Table 6 Effect of chitosan and their derivatives on DTH assay (Mean±SE，16d) Group

Dose (mg/kg)

Number (n)

paw thickness (mm)

Con

0

12

0.505±0.053

Levison

20

11

0.527±0.053

CTS

100

12

0.615±0.037

WCOS

33

12

0.596±0.059

100

12

0.684±0.056

333

12

0.855±0.066 ***

33

12

0.568±0.073

100

12

0.653±0.079

333

12

0.602±0.048

33

12

0.644±0.036

100

12

0.585±0.047

333

12

0.562±0.057

YCOS

ZCOS

20   

*** p <0.001, compared with control group.     Table 7 Effects of chitosan and their derivatives at different doses on index of spleen and thymus and carbon particle clearance rate (Mean±SE，16d)

Group Dose(mg/kg)

Number

Spleen index

Thymus index

Phagocytic

(n)

(mg/g)

(mg/g)

indices (α)

Con

0

12

3.14±0.17

2.46±0.12

2.54±0.29

Levison

20

12

2.97±0.27

3.08±0.33

3.82±0.20*

CTS

100

12

3.06±0.18

2.73±0.27

3.23±0.24

WCOS

33

12

2.99±0.28

2.59±0.26

3.43±0.31

100

10

4.54±0.26*

2.53±0.22

4.23±0.25*

333

13

2.56±0.20

1.92±0.33

4.20±0.32*

33

11

4.43±0.55

2.59±0.30

3.64±0.25

100

13

2.99±0.17

2.15±0.20

3.44±0.19

333

10

3.28±0.37

2.92±0.19

4.49±0.50**

33

10

2.76±0.11

2.38±0.19

3.56±0.65

100

12

2.68±0.13

2.61±0.22

4.89±0.40***

333

12

3.23±0.22

2.90±0.23

3.68±0.41

YCOS

ZCOS

* p < 0.05 , ** p < 0.01,*** p < 0.001, compared with control group.  

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