Anti-cancer Activity Of The Purified Extract Glycoprotein With Salamander Skin Mucus

The Chinese Giant Salamander(Andrias davidianus) is a large amphibia, caudata, implicit gill salamanders, which is a class 2 nationally protected animal and is regarded as living fossil. Giant Salamander has two types of glands: mucous gland and granular gland. Mucus gland could release glycoprotein, which could become mucus after combined with water. It coves the free surface of epithelium and forms a natural protective barrier. There is a special skin gland that secretes a kind of scent of white mucus. It contains antibacterial peptide, glycoprotein and other pharmacological active substances that reveal many complex functions such as, to widely antibacterial, antioxidant, anti-fatigue, antitumor etc. it plays a very significant role in these fields.This paper take research on the skin mucous Giant Salamander as the object. By using the method of extraction and chromatography different preparationn, research was carried out on the appearance of skin mucous glycoprotein, and uses the MTT colorimetric method for detecting their antitumor activity in vitro situation. The main results were as follows:(1) The analysis on components of skin mucus. Through the composition detection test of skin mucous in sugar, fat, protein, it showed that the total sugar content in the appearance of mucus was 3.69%. Crude fat content is 4.25%, which contains 11 kinds of fatty acids, unsaturated fatty acids oleic acid, linoleic acid and linolenic acid content is higher, are 27.73%, 9.80% and 27.73% respectively; In saturated fatty acids with palmitic acid, myristic acid, stearic acid content is higher, 21.53%, 5.92% and 3.61% respectively.Total amino acids content reaches 83.97%, containing 17 kinds of amino acid composition, 8 kinds of essential amino acids for human beings. Except for no data the tryptophan from hydrolysis, we have detected the rest 7 kinds of amino acid content accounted for 33.96% of total amino acids.Through to the appearance of mucus in the volatile gas composition analysis, detection of 10 kinds of volatile substances, including dimethyl disulfide ether(C2H6S2) and eight kinds of lipid compounds, and dimethyl disulfide ether as the main cause of special smell.(2) The analysis on appearance of mucous glycoprotein extraction and separation and purification.Based on the investigation to the alkali extraction process of the steps, and on the premise of glycoprotein antitumor biological activity, and finally determine the appearance of mucous glycoprotein extraction process is: 0.4 mol/L Na OH solution, and solid-liquid ratio in 25, under 45 ℃, 2 h. Extract to 4000 r/min, 15 min, the centrifugal supernatant fluid adding anhydrous ethyl alcohol sink to the concentration of 76%, the p H to 7.0 or so, in 4 ℃ refrigerator let stand for 8 h; To remove supernatant liquid centrifugally, we wash glycoprotein precipitation with ethanol after three times, vacuum drying glycoprotein crude products.For raw Giant Salamander mucous glycoprotein with DEAE-52 anion exchange column chromatography separation, collect the sample peak, concentrated again with Sephadex G-100 gel column chromatography separation and purification.The research end up with a relatively pure glycoprotein component, by SDS protein electrophoresis tests, it contains a single glycoprotein component, its molecular weight is about 30 k Da.(3) The analysis on the appearance of mucous glycoprotein composition analysis.This study anglicizes appearance of mucous glycoprotein monosaccharides composition and amino acid composition by high performanceed liquid chromatography(HPLC) and automatic amino acid analyzer.Eventually determine the appearance of mucous glycoprotein contains six kinds of monosaccharide components, the molar ratio of mannose(Man) : glucose aldehyde(Glc UA) : galacturonic acid(Gal UA) : n-acetyl glucosamine(Glc N) : glucose(Glc) : galactose(Gal) = 1.71:4.12:1.09:1.55:1.00:2.09. All together the study detected 17 kinds of amino acid, total amino acid content is 61.86%. Showed total amino acid content is 61.86%, the 17 kinds of amino acids, glutamic acid, proline, glycine and aspartic acid content is higher, 7.20%, 6.39%, 5.62% and 5.43% respectively.(4) The analysis on the appearance of mucous glycoprotein antitumor activity in vitro study.Determined by MTT experiment in vitro tests, Giant Salamander mucus glycoproteins fight against human lung adenocarcinoma A549 cancer cells.Role when the concentration of glycoprotein is 40ug/m L, 48 h, alkali extraction glycoprotein, DEAE-52 chromatography glycoprotein chromatography and Sephadex G-100 glycoprotein of A549 cells reached the highest inhibition rate of 88.43%, 91.34% and 92.32% respectively. With glycoprotein sample concentration from 1, 10, 20, 40 ug/m L, different sources of mucus glycoprotein on A549 cell inhibitory effect from high to low respectively: Sephadex G-100 chromatography glycoprotein > DEAE-52 chromatography glycoprotein > alkali extraction of glycoprotein. With the extension of incubation time, the appearance of mucous glycoprotein on A549 cell inhibitory effect obviously increases, the inhibition rate in a certain concentration range time dependence. Mucus glycoprotein compared with positive control drug, on A549 cell proliferation inhibition extremely significant difference(p < 0.01).Using inverted optical microscope to examine the mucous glycoprotein function before and after 48 hours, focusing on the shape change which giant salamander against lung adenocarcinoma A549 cancer cells.Results show that under the light microscopy are cells decreased significantly, the cells become smaller, appear fold, turn round, and less correlated with the surrounding cells, some cells fall off from the bottom wall such as obviously, morphological changes.