| Objective In this paper, by isoelectric precipitation method extracted natural seaweed pigment glycoprotein(NSPG) from Eucheuma, determined it’s structure by IR、UV、Liquid-mass spectrometry and gel permeation chromatography. By adopting the method of cell culture in vivo and in vitro, observe the effect of NSPG on the inhibition of hepatoma cells.Methods1Preparation and analysis of NSPG1.1NSPG Preparation:Eucheuma was cut into0.5-1.0cm long of strips, placed in a solution of hydrochloric acid pH3.0, the collected filtrate was adjusted to pH4.5, and then adding3volumes of95%ethanol, stirred, allowed to stand overnight, precipitated and filtered to obtain the precipitate on the filter paper part, and the precipitate portion were dried over anhydrous ethanol, washed with acetone, dried at room temperature, obtained NSPG1.2NSPG Analysis:1.2.1NSPG polysaccharide and protein content determination:Glucose as the standard material, using sulfuric acid-phenol method for the determination of glucose content. Bovine serum albumin as a standard material, with Coomassie brilliant blue protein content were determined.1.2.2NSPG structural analysis:UV spectra and IR spectra were determined polysaccharides and proteins characteristic absorption peaks.According to β-elimination reaction and liquid chromatography-mass spectrometry results determined glycosidic bond type.1.2.3NSPG molecular weight determination:The molecular weight of NSPG was determined by gel chromatography.1.2.4Sensory indicators:Observed the shape, color and other indicators.2NSPG on the inhibition of liver cancer2.1In vitro experiment:Different concentrations of NSPG (100mg/L50mg/L、10mg/L) were cultured with H22hepatocarcinoma cells of mice and HepG2human hepatoma cells. Without NSPG was set as a separate control group, and the cell proliferation was determined by MTT assay.2.2In vivo experiment:Under sterile conditions, the H22passage section of the mice ascites liver cancer was inoculation to the weighing18-20g50healthy Kunming mice, which were inoculated subcutaneously at right axilla0.2ml. All of the animals were randomly divided into five groups, which were control group、high、medium and low doses of natural seaweed pigment glycoprotein group and cyclophosphamide group with half gender mice in each group. Mice in high, medium and low dose group were given by gavages concentration of100、50and10mg/kg natural seaweed pigment glycoprotein aqueous, the cylophosphamide group were injected20mg/kg every the other day, and control group were treated with saline, once a day for10days. The day after the last dose the mice were weighed'abstract mice eyeball blood, serum was separated'the tumor weights spleen weight and thymus weight were examined by electronic scale'stripping tumor tissue to be seized.2.2.1Tumor index and inhibitory rate:The tumor weight and body weight were examined by electronic scale and the tumor index and inhibitory rate were calculated.2.2.2Thymus index and spleen index:The thymus weight and spleen weight were examined by electronic scale and indexes were calculated.2.2.3Serum levels of TNF-α:The serum TNF-α was measured by enzyme linked immunosorbent assay. 2.2.4The proliferative activity:Determined by MTT proliferation of tumor cells.2.2.5Protein expression:The level of PCNA、Bcl-2、Caspase-3and Bax protein was measured by immunohistochemistry method.Results1Preparation and analysis of NSPG1.1NSPG Preparation:Yield was8.3%.1.2NSPG Determination:Polysaccharide content of93.3%, the protein content of2.04%.1.3NSPG structural analysis:In the UV spectrum, at195nm appears polysaccharide characteristic peaks, Its absorption value is1.834; at280nm appears protein’s absorption value is0.417, but the absorption peak is not obvious, indicating that low levels of protein. In the IR spectrum, showing characteristic absorption of an amide bond、sulfate groups and sulfate group substitution positions、pyranose glycosidic bond characteristic absorption peak, these proved that NSPG is a glycoprotein. After β-elimination reaction the UV absorption and amino acid composition have changed, indicating that there is O-connected glycosidic bond. In the mass spectrum, the difference between each peak is115, which is the molecular weight of aspartic acid. Description NSPG has N-linked glycosidic bond too.1.4NSPG molecular weight determination:GPC analysis showed that NSPG relative molecular weight is213.8kDa.2NSPG inhibition on H22hepatoma2.1In vitro experiment results:The proliferative activity of cultured cells in vitro in100mg/LNSPG group were0.40±0.08, which were significantly lower than the tumor control group (P<0.05), and those in10mg/L and50mg/L NSPG group were0.52±0.09and0.45±0.05respectively. Indicates with increasing doses of NSPG, proliferation activity of H22hepatoma cells was decreased (P<0.05),and that a certain dose of NSPG has inhibition effect on proliferation activity of H22hepatoma cells.2.2In vivo experiment results:2.2.1Tumor index and inhibitory rate:The average of tumor weight in high dose group and tumor control group were0.31±0.05g and0.55±0.23g respectively, the differences were statistically significant (P<0.05).2.2.2Thymus index and spleen index:High-, middle-and low-dose group NSPG thymus index and spleen index compared with the control group, no significant differences (P>0.05), but higher than the tumor control group (P<0.01).2.2.3Serum levels of TNF-α The serum TNF-a concentration of the high dose NSPG group were92.17±4.01(pg/ml), significantly lower than that of the tumor control (p<0.05).2.2.4The proliferative activity:The proliferation activity of the tumor control group was1.14±0.03, significantly higher than that of the high-dose NSPG group (P<0.05).2.2.5Expression Results:The PCNA positive expression rate in tumor control group were72.78%, and those in high dose group were28.32%, differences were significant (P<0.05). The positive expression level of Bax and Bcl-2protein in high-dose group were38.10%and16.78%, respectively, and those of the tumor control group were4.68%and65.16%, respectively. There were significant differences between the groups (P<0.05). The A value of Caspase-3of the high-dose NSPG group was0.53±0.09, significantly higher than that of the tumor control group (P<0.05).Conclusion1. Established the method of extraction of NSPG from Eucheuma. NSPG relative molecular weight is213.8kDa.NSPG polysaccharide content was93.3%, protein content was2.04%. There was O-connected glycosidic bond, and has N-linked glycosidic bond too. Yield was8.3%. 2. NSPG not only for H22hepatoma cells and human liver cancer cell proliferation with a certain degree of inhibition in vitro, on H22hepatoma cells also inhibited in vivo, indicating NSPG can inhibit liver cancer cell proliferation activity.3. NSPG could reduce the expression level of Bcl-2and PCNA protein on H22hepatoma tissue; promote the expression of Bax and Caspase-3protein on H22hepatoma tissue. Description by inhibiting the proliferation and promoting the apoptosis inhibit the growth of liver cancer.4. NSPG suppress abnormal elevation of serum TNF-α, further evidence that NSPG inhibiting the growth of liver cancer.5. Although cyclophosphamide was inhibited on H22cells, but it can damage the spleen and thymus, which led to the decline of immune function. However, NSPG not only inhibit the growth of H22hepatoma cells, but also has a protective effect on the spleen and thymus. NSPG can inhibit growth of H22hepatoma tumor on mice, which provide theoretical basis on the development of Eucheuma and prevention and treatment of liver diseases. |
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