Study On Structure, Antitumor And Immunological Activities Of The Polysaccharide From An Endophytic Fungus Of Crocus Sativus L.

As a natural biological macromolecules, fungal polysaccharide have increased more and more attention in the biological and medicine and daily chemical industry. Crocus sativus L.(CSL) is useful biologically active ingredients for pharmaceutical use such as antitumor, immunoregulation, anti-hypertensive, anti-depressant, anti-inflammatory, and relaxant activities. It has been reported that CSL has the strong cytotoxicity on HeLa cell and enhances the activity of macrophage. But the pharmaceutical ingredients of CSL are just collected from the stigma andits product quality is difficult to control when CSL is traditionally cultivated. The yield of CSL can’t meet the need of market. Based on the preliminary work, the polysaccharide isolated from the endophytic fungus(CSL-27) showed strong antioxidant activities and moderated antitumor activities. Thus, we systematically study the extraction, purification, structure and biological activity of this polysaccharide in this paper. The main results are as follows:Part 1: Response surface methodology with Box-Behnken design was used to optimize the extraction procedure of polysaccharide from the endophytic fungus(CSL-27). Firstly, the single-factor experiment was carried out. The yield of polysaccharide was used as an index and precipitation time, precipitation temperature, pH and ratio(95%ethanol to fermented broth) were investigated respectively. The precipitation time(16 h), precipitation temperature(4 °C), pH(7) and ratio(4:1) were set as the central point on three level in the Box-Behnken design. Combined with the analysis of response surface methodology experiment, the optimized conditions were as follows: precipitation time(16 h), precipitation temperature(3.7 °C), pH(7.2) and ratio 5:1(L/L). Under these conditions, the experimental yield of EPS was 162 μg/L, which was close to the predicted yield value, 165 μg/L. The optimized technology can effectively improve the yield of polysaccharide, which provides the basis for further study.Part 2: The column chromatography was used to purify the polysaccharide from CSL-27. The physical and chemical properties of polysaccharide were characterized by the chemical and instrumental analysis and the structure of EPS-2 was determined. The polysaccharide was conducted with the following steps: deproteinizing with Sevag method, decoloring by resins and dialyzing and freeze-drying. Then the EPS was isolated by DEAE-52 ion-exchange column chromatograph, which the polysaccharide EPS-1 and EPS-2 were gathered. They were identified to be a homogeneous polysaccharide due to a single symmetrical peak in the elution curve of SephadexG-75 column chromatography. Then the sugar content of EPS-1 and EPS-2 were 48.60% and 28.89% respectively, which was standardized by glucose. The results of high performance liquid gel exclusion chromatographic showed that the EPS-1 and EPS-2 were both homogeneous polysaccharides for the single chromatographic peak and their molecular weights were 29409 and 40427 respectively. The results of high performance liquid chromatography determination displayed that the monosaccharide composition of EPS-1 was mannose, glucose, xylose and arabinose, its ratio was 2.3:1.0:1.1:1.4; the monosaccharide composition of EPS-2 was mannose, glucose and arabinose, its ratio was 9.2:6.4:1.0. The Ultraviolet spectrum proved that the EPS-2 did not contain proteins or nucleic acids which was no absorption at 260 nm and 280 nm. The Infrared spectra of EPS-2 revealed multiple characteristic absorption peaks at 3367 cm-1, 2939 cm-1, 1131 cm-1 and 1056 cm-1 and it might be composed of β-D-pyranoid ring and α-gycosidic linkage. The surface structure of EPS-2 was uneven and folding gap which was analyzed by scanning electron microscope. The result of GC-MS showed that four sugar residue substrate period contained(1â†'6) linked β-D-glucopyranose,(1â†'2,4) linked α-D-mannopyranose terminal α-L-arabinofuranose and terminal α-D-mannopyranose and the molar ratio of was 6.25:1.00:4.47:6.32. Finally, the nuclear magnetic resonance spectrum was used to determine the structure of EPS-2 which comprised a backbone of(1â†'6) linked β-D-glucopyranose(1â†'2,4) linked α-D-mannopyranose with branches consisting of terminal α-L-arabinofuranose and terminal α-D-mannopyranose.Part 3: The assay of MTT method was used to in vitro evaluate the anti-proliferative activity and immunological activity of EPS-2. The human erythroleukemia K562 cells, human lung adenocarcinoma A549 cells, human promyelocytic leukemia HL-60 cells and human cervical carcinoma HeLa cells was used to evaluate the anti-proliferative activity of EPS-2. The result of antitumor experiment showed that at the concentration rang of 0~5 mg/m L, the EPS-2 exhibited little inhibition on K562, HL-60 and A549 cells. However, the EPS-2 inhibited the moderated proliferation of HeLa cells at 0.5 mg/mL. Compared to the prophase work, the antitumor activities had reduced for the lack of pigment and EPS-1 from crude polysaccharide. The macrophages RAW264.7 played an important role in the immune response, so the promoting proliferation activity of EPS-2 for macrophage was used to evaluate the immunological activity. The result showed that at the concentration rang of 0~5 mg/mL, the EPS-2 exhibited little proliferation activity on mice abdominal cavity macrophage RAW264.7 cells. The EPS-2 can’t combined with the receptors on the surface of the macrophage RAW264.7 which can’t promote the secretion of inflammatory factors to immunoregulation.