Chunhui Liu

Isolation, structural characterization and immunological activity of an exopolysaccharide produced by Bacillus licheniformis 8-37-0-1

A strain of Bacillus licheniformis 8-37-0-1 with high exopolysaccharide (EPS) production ability was isolated and identified based on morphological and physiological characteristics and phylogenetic analysis of 16S rDNA sequences. A new type of EPS was isolated from the strain fermentation broth by enzymolysis, isopropanol precipitation, anion-exchange, and gel-filtration chromatography. The new EPS was determined as homogeneous, with a molecular weight of 2.826 x 10(4), as determined by High-Performance Size-Exclusion Chromatography Multi-Angle Laser Light Scattering analysis. Its structural characteristics were investigated and elucidated by methylation analysis, partial acid hydrolysis, gas-liquid chromatography mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance spectroscopy. Based on obtained data, the EPS was found to be a levan containing a (2-->6)-linked backbone with a single beta-d-fructose at the C-1 position every seven residue, on average, along the main chain. Preliminary in vitro tests revealed that EPS could significantly stimulate the proliferation of spleen lymphocyte.

Structural characterisation and antimutagenic activity of a novel polysaccharide isolated from Sepiella maindroni ink

A new heteropolysaccharide, named as SIP, was isolated from the ink of cuttlefish, Sepiella maindroni, by enzymolysis, anion-exchange and gel-permeation chromatography and tested for its antimutagenic activity. It was homogeneous with a molecular weight of 1.13×10(4)Da by HPSEC-MALLS analysis. SIP contained glucuronic acid, mannose, N-acetylgalactosamine, and fucose in a molar ratio of 1:1:2:2. Its structural characteristics were investigated and elucidated by methylation analysis, GLC-MS, and NMR ((1)H, (13)C, H-H COSY, HMQC, HMBC, TOCSY and NOESY). The hexasaccharide repeating unit of SIP was found to be a backbone composed of fucose, N-acetylgalactosamine and mannose in a molar ratio of 2:2:1, and with a single branch of glucuronic acid at the C-3 position of mannose. According to the micronucleus test, SIP could significantly reduce the frequency of micronucleated cells in polychromatic erythrocytes and reticulocytes induced by cyclophosphamide in tumor-bearing mice, which revealed that SIP presented strong antimutagenic activity.

Anti-metastatic and anti-angiogenic activities of sulfated polysaccharide of Sepiella maindroni ink.

A previous study demonstrated that SIP-SII, a sulfated Sepiella maindroni ink polysaccharide, suppressed the invasion and migration of cancer cells via the inhibition of the proteolytic activity of matrix metalloproteinase-2 (MMP-2). Therefore, this study investigated the anti-metastatic effect of SIP-SII in vivo. SIP-SII (15 and 30 mg/kg d) markedly decreased B16F10 pulmonary metastasis in mice models by 85.9% and 88.0%, respectively. Immunohistochemistry showed that SIP-SII decreased the expression of the intercellular adhesion molecule 1 (ICAM-1) and basic fibroblast growth factor (bFGF) in lung metastasis nodules. In addition, SIP-SII inhibited neovascularization in chick chorioallantoic membrane assay at 0.08-2 mg/mL. In the in vitro experiments, SIP-SII (0.8-500 μg/mL) significantly decreased the protein and mRNA expression of ICAM-1 and bFGF in SKOV3 and EA.hy926 cells, respectively. These results suggested that SIP-SII might suppress melanoma metastasis via the inhibition of the tumor adhesion mediated by ICAM-1 and the angiogenesis mediated by bFGF, as well as resulting in depression of the invasion and migration of carcinoma cells.

Isolation, structural characterization and neurotrophic activity of a polysaccharide from Phellinus ribis

A new polysaccharide named PRG was isolated from the fruiting bodies of Phellinus ribis by hot water extraction, ethanol precipitation, anion-exchange and gel-filtration chromatography. PRG was homogeneous, with a molecular weight of 5.16 × 10(3)Da, as determined by high-performance size-exclusion chromatography-multiangle laser light scattering analysis. Its structural characteristics were investigated and elucidated by methylation analysis, partial acid hydrolysis, gas-liquid chromatography mass spectrometry, Fourier transform infrared and nuclear magnetic resonance spectroscopy. Based on obtained data, PRG was found to be a β-d-glucan containing a (1 → 3)-linked backbone, with a branch of two (1 → 6)-linked and one terminal glucoses substituting at the C-6 position every three residues, along the main chain. PRG exhibited neurotrophic activity, which significantly promoted the neurite outgrowth of the nerve growth factor-stimulated PC12 cells, suggesting that it might be a potential candidate for the treatment of neurodegenerative diseases.

Anti-tumor activity and the mechanism of SIP-S: A sulfated polysaccharide with anti-metastatic effect

Our previous studies demonstrated that SIP-S had anti-metastatic activity and inhibited the growth of metastatic foci. Here we report the anti-tumor and immunoregulatory potential of SIP-S. SIP-S could significantly inhibit tumor growth in S180-bearing mice, and the inhibition rates was 43.7% at 30 mg/kg d. Besides, SIP-S could improve the thymus and spleen indices of S180-bearing mice and the mice treated with CTX. The combination of SIP-S (15 mg/kg d) with CTX (12.5 mg/kg d) showed higher anti-tumor potency than CTX (25 mg/kg d) alone. These results indicated that SIP-S had immunoenhancing and anticancer activity, and the immunoenhancing activity might be one mechanism for its anti-tumor activity. Flow cytometry results showed that SIP-S could induce tumor cells apoptosis. Western blot analysis indicated that SIP-S could upregulate the expression of pro-apoptotic proteins, caspase-3, -8, -9 and Bax, and downregulate the expression of anti-apoptotic protein PARP-1 in tumor cells in a dose-dependent manner. In summary, SIP-S has anti-tumor activity, which may be associated with its immunostimulating and pro-apoptotic activity.

Protective effects of low molecular weight chondroitin sulfate on amyloid beta (Aβ)-induced damage in vitro and in vivo.

In the present study, we investigated the effects of low molecular weight chondroitin sulfate (LMWCS) on amyloid beta (Aβ)-induced neurotoxicity in vitro and in vivo. The in vitro results showed that LMWCS blocked Aβ25-35-induced cell viability loss and apoptosis, decreased intracellular calcium concentration, reactive oxygen species (ROS) levels, the mitochondrial membrane potential (MMP) depolarization, and the protein expression of Caspase-3. During in vivo experiments, LMWCS improved the cognitive impairment induced by Aβ1-40, increased the level of choline acetyltransferase (ChAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and decreased the level of malondialdehyde (MDA) and acetylcholinesterase (AChE) in the mouse brain. Moreover, LMWCS decreased the density of pyramidal cells of CA1 regions, and suppressed the protein expression of Bax/Bcl-2 and Caspase-3, -9 in the hippocampus of mice. In conclusion, LMWCS possessed neuroprotective properties against toxic effects induced by Aβ peptides both in vitro and in vivo, which might be related to anti-apoptotic activity. LMWCS might be a useful preventive and therapeutic compound for Alzheimers disease.

Characterization and Secondary Structure Analysis of Endostatin Covalently Modified by Polyethylene Glycol and Low Molecular Weight Heparin

Endostatin (ES), as an angiogenesis inhibitor, has been approved by the State Food and Drug Administration (SFDA) in China for the treatment of patients with non-small-cell lung cancer. However, as a protein drug, there are a lot of obstacles on its clinical application, such as need of high dose to maintain its efficacy, expensive and poor stability, etc and limits its clinical use. In order to overcome these shortcomings, we chemically modified ES by polyethylene glycol and low molecular weight heparin (LMWH), respectively. The changes of the secondary structure of the modified products were studied by Fourier transform infrared spectroscopy and Circular dichroism spectra to obtain better ES derivatives. Our study demonstrated that the modified products have a better heat tolerance than ES towards. The result of secondary structure analysis suggests the percentage of beta-turn in whole protein is an important factor on the activity and heat stability and ES modified by LMWH can maintain higher activity and its secondary structure.

Augmented inhibitory effect of superoxide dismutase on superoxide anion release from macrophages by chemical modification with polysaccharide and attenuation effects on radiation-induced inflammatory cytokine expression in vitro

To improve the ability of superoxide dismutase (SOD) to suppress reactive oxygen species (ROS)-mediated injury, chemically modified derivatives of SOD with N,N,N-trimethyl chitosan chloride (TMC) and heparin, cationized SOD (TMC-SOD), and anionized SOD (heparin-SOD) were designed and prepared. In this study, the inhibitory effect of TMC-SOD and heparin-SOD on superoxide anion release from macrophages was studied in vitro. Both TMC-SOD and heparin-SOD exhibited excellent inhibitory effects on superoxide anion release from macrophages, and the effects of TMC-SOD surpassed those of native SOD and heparin-SOD. The effects of TMC-SOD and heparin-SOD on inflammatory cytokine expression in vitro were also evaluated. The results showed that both TMC-SOD and heparin-SOD could significantly lower the levels of transforming growth factor-β1 (TGF-β1) and interleukine-1β (IL-1β) expressed by irradiated 3T3 fibroblasts. These results demonstrated that cationic polysaccharide or anionic polysaccharide SOD derivatives might be useful in the prevention and treatment of ROS-mediated inflammatory diseases. This study also demonstrated that chemical modification of SOD, especially cationization, greatly enhanced SOD’s intracellular delivery and, consequently, produced a significant protective effect against ROS-mediated injury.

Enhanced anti-angiogenesis and anti-tumor activity of endostatin by chemical modification with polyethylene glycol and low molecular weight heparin.

Endostatin (ES), a potent endogenous angiogenesis inhibitor found in 1997 by OReilly, can effectively inhibit angiogenesis, inhibit the growth and metastasis of tumors. ES can also decrease drug resistance in long term and repeated treatment when it is used in combination with other chemotherapeutic agents. But there are still lots of obstacles on its clinical application, such as the need of a high dose to maintain its efficacy short half-life, poor stability, expensive, and some other shortcomings just like other protein drugs. Chemical modification on ES by polyethylene glycol (PEG) and low molecular weight heparin (LMWH) were successfully carried out in order to obtain a better ES derivative. Several classic experimental models were employed to study the bioactivity of ES and modified ES, including chicken chorioallantoic membrane (CAM) assay, corneal neovascularization (CNV) assay and Sarcoma 180 tumor bearing mice assay. The results showed that PEG-ES and LMWH-ES had better anti-angiogenesis and anti-tumor activity than ES, which indicates that LMWH was also a good protein modifier.

Simultaneous analysis of heparosan oligosaccharides by isocratic liquid chromatography with charged aerosol detection/mass spectrometry.

Uncovering the biological roles of heparosan oligosaccharides requires a simple and robust method for their separation and identification. We reported on systematic investigations of the retention behaviors of synthetic heparosan oligosaccharides on porous graphitic carbon (PGC) column by HPLC with charged aerosol detection. Oligosaccharides were strongly retained by PGC material in water-acetonitrile mobile phase, and eluted by trifluoroacetic acid occurring as narrow peaks. Addition of small fraction of methanol led to better selectivity of PGC to oligosaccharides than acetonitrile modifier alone, presumably, resulting from displacement of methanol to give different chemical environment at the PGC surface. Vant-Hoff plots demonstrated that retention behaviors highly depended on the column temperature and oligosaccharide moieties. By implementing the optimal MeOH content and temperature, a novel isocratic elution method was successfully developed for baseline resolution and identification of seven heparosan oligosaccharides using PGC-HPLC-CAD/MS. This approach allows for rapid analysis of heparosan oligosaccharides from various sources.