Preparation Of Polysaccharide From Porphyridium Cruentum And Its Biological Activities

The Porphyridiun cruentum (P.cruentum) are the only unicellular marine microalga in Rhodophyta, which can synthesize and secrete extracellular polysaccharides (EPS) into culture liquid when cells are grown in steady-state culture. The yield of EPS usually correlates with the culture condition of microalga. There have been made some progress about cultivation and bioactive compounds of P. cruentum. However, the researches about the molecular modification of P. cruentum polysaccharide, especially the relationship between structure and biological activities are still rare. In present work, the yield of EPS was improved effectively by ecology control and optimization of technical condition. Based on analysis of physicochemical properties and chemical structure of polysaccharide, different molecular and different sulfate content derivatives of polysaccharide extracted from P. cruentum were prepard. Thereafter, the relationship between different properties polysaccharide and the antioxidant activity, anti-tumor activity and immunomodulation activity were investigated. The results were summarized as follows:1 The culture conditions for improvement of EPS production(1) In order to probe the effect of nutrient salts on polysaccharide yield of P. cruentum, several important inorganic compounds such as nitrogen, phosphor, sulfate, metal ion and plant growth hormone were investigated. The optimal cultural medium (OCMâ…¡) for increasing EPS yield were obtained with NaHCO₃ 3.5 g, KH₂PO₄ 20 mg, NaNO₃ 2 g, Na₂SO₄ 20 mg, FeCl₂·6H_O 10μM, and MnCl₂·4H₂O 0.2μM, V_B1 0.9 mg, V_B12 2.7μg, sterile seawater 1000 mL. The maximal EPS production achieved as 672 mg/mL after 15 d cultivation in OCMâ…¡medium, which increased 1.79 times and 1.62 times compared with that cultivated in 2/f and Koch medium.(2) In order to investigate light quality on yield of EPS, the P. cruentum were cultivated in flat plate photobioreactors (FPPBR) developed by ourselves and different light illumination intensity, light/dark cycle, light spectrum and light pathway of reactor were designed. The optimum light illumination conditions for maximal EPS yield were photon flux density 80μE·m^-2s^-1, light/dark cycle 18:6, natural light or yellow light, and the maximal yield of polysaccharide was 0.96 g/L. The maximum EPS yield was observed as 1.13 g/L in FPPBR with light-pathway of 30 mm, but the maximal areal output rate of EPS achieved as 5.8 g /rn²/d in FPPBR with light-pathway of 50 mm. (3) To probe the effects of renewal regime on the production of polysaccharides, P. cruentum were cultured semicontinuously in flat plate photobioreactor. Uniform design was used to optimize renewal conditions. Quadratic mathematic models related to output rate, total recovery yield of biomass and polysaccharides were set up to clarify the influence of individual factors and their interactions. According to the mathematic models, the optimal semi-continuous condition for total yield of EPS was NaNO₃ 3.5 g/L, renewal rate 27%, renewal period 2.91 d. With the optimal renewal regime, the maximal total recovery yields and average production of EPS achieved at 29.4 g and 1.96 g/L, respectively, which was 2.35 and 1.57 times higher than that of batch cultivation.2 Preparation of different EPSs and analysis of structure and properties(1) The EPSs were isolated and purified by DE52 hydronium exchange chromatography column and Sephacryl S-400 gel chromatography column, then the homogeneous water-solubility polysaccharides were obtained. Analysis of physicochemical properties showed that the apparent molecular weight (MW) of EPS was about 2918 kDa, sulfate content was 14.63%, glucurouic acid content was 7.8%. Rheological behavior showed that the aqueous solution of EPS was typical pseudoplastic fluid, and the viscosity of polysaccharide could not change with temperature and heating time. GC analysis showed that the monosaccharide components of EPS were mainly composed of with xylose, glucose and galactose, and molar ratio of each sugar is 2.96: 1.25: 3.06. Results of chemical and spectral analysis showed that the main linkage wasβ-(1→3), and there were a few of 1→4 and 1→6 linkages. A large of galactoses exsited at the end or branch of sugar chain, but xylose and glucose were distributed specially close to main chain. So it can be concluded that the possible repeating unit of EPS could be shown as: (2) The molecular weights of degraded polysaccharides prepared by hermetical microwave were 6553 Da, 60.66 kDa and 56.26 kDa, respectively. Oxidative degradation with H₂O₂ under ultrasonic wave were used to degrade EPS from 2918 kDa to 203.6 kDa, 606.6 kDa, 802.6 kDa, 903.3 kDa and 1002 kDa. The EPS derivatives with sulfate content of 31.0%, 31.5%,47.5% and 42.4% were prepared by controlling different reaction temperature. The physicochemical properties before and after degradation had no obvious changes, and the water-solubility of derivate EPS increased obviously.3 Biological activities of EPS(1) The antioxidant properties of EPS were evaluated by determining the scavenging ability of free radicals, inhibitory effects on Iipid peroxidation in mouse liver homogenates and hemolysis of mouse erythrocytes. The results showed that all degraded polysaccharide fragments had strong scavenging effects on hydroxyl-radical (·OH), Superoxide-radical (O₂^-·) and 1, 1-Diphenyl-2-picrylhydrazyl (DPPH·). Besides, the scavenging activities of EPS improved with an increase of samples concentration. All degraded polysaccharides clearly inhibited Iipid peroxidation induced by Fe²⁺/ascorbic acid in mouse liver homogenates, and markedly inhibited H₂O₂-induced hemolysis of mouse red blood cells (RBCs) in a dose-dependent manner.MW of EPS had significant effect on antioxidant ability. High-molecular-weight EPS before degradation had no obvious antioxidant activity, but the antioxidant activities increased with the decrease of MW. The 6.55 kDa fragment had stronger antioxidant activity than the others. The antioxidant activity of sulfate derivatives of EPS improved distinctly, and the IC₅₀ values of scavenging·OH and O₂^-·were 3.92 mg/mL and 0.4mg/mL,respectively. With an increase of sulfate content, the scavenging effects of polysaccharide derivatives increased, but the difference between them was not obvious significance.(2) The inhibitory effects of EPS on tumor cells in vitro were determined by method of SRB. The results showed that high-MW EPSs had no anti-tumor activity. The 6.55 kDa-fragment and 256 kDa-fragment had some inhibitory effects on lung adenocarinoma cells (A549), laryngeal epidermal cancer cells (Hep-2) and hepatoma cells (SMMC7721), but the effects were not obvious.The effects of EPS on Sarcoma 180 (S180) tumor growth were observed after different doses of EPS were administrated by gastric perfusion to S180-bearing mice. The growth of S180 tumor was significantly inhibited by EPS, and the growth inhibitory rate were 53.3%,47.5% and 40.5%, repectively at the dose of 200, 100 and 50 mg/kg/d. The effects of EPS on immune system in vivo indicated that EPS could increase remarkably spleen and thymus index in S180-bearing mice, and could also stimulate significantly spleen lymphocyte. The pro- liferation indexes of lymphocyte were 2.41 and 2.15 compared with model groups and positive control (CTX) groups, respectively.(3) The immunomodulating assay in vitro showed EPS could improve production of NO of mouse macrophage, boost up neutral red uptake, stimulate mouse macrophage and spleen lymphocyte proliferation at the dose range of 25-200μg/mL in a dosage-dependent way. The immunomodulating ability between different molecular weight EPS had significant differences,and 6.55 kda-fragment had strongest activity. Sulfate content of EPS derivatives can also affect the immunomodulation activities significantly. With an increase of sulfate content, the immuno-accelerating effects enhanced, so the derivative with sulfate content of 48% had the strongest activity. However, the best effect on macrophage proliferation was observed at fragment with sulfate content of 32%.