Puirifcation Of Glycoprotein From Camellia Oleifera Abel Seeds And Its Physiological Activities

Camellia oleifera Abel is a typical woody oilplant in China, and notable as an importantsource of edible oil obtained from its seeds. Camellia oleifera is also known as multipurposeplant applied as medicament, chemical, fodder, ornamental. At present, the single purpose ofCamellia oleifera is to squeeze oil, its medicinal value recorded by Chinese traditionalherbalist is ignored. The current issues, such as Camellia oleifera and its by-product with lowdegree of deep processing and comprehensive utilization, have limited development ofCamellia oleifera industry. In this study, we studied the extraction, purification, physical andchemical properties and structure feature of glycoprotein (COGP2a) from Camellia oleifera.The anticancer and antioxidant activity of COGP2a both in vitro and in vivo and themechanism of apoptosis on HepG2tumor cells were also investigated. These study resultscould improve the utilization level of Camellia oleifera resources, promote the comprehensivedevelopment of Camellia oleifera and rich active glycoprotein type and function, which hasimportant important theoretical and realistic significance.The extraction conditions of two kinds extraction methods, the buffer solution and waterextraction method, were optimized by response surface method, and establish the regressionmodel, the equation analog effect was good. Under the optimal conditions of buffer solutionextraction, the protein yield was8.76%, sugar yield was10.14%. Under the optimalconditions of water extraction, the protein yield was8.96%, sugar yield was17.05%.Antitumor activities of the two crude glycoproteins prepared by buffer solution and waterextraction method at different concentrations were examined by MTT assay. The resultsshowed that the two crude glycoproteins exhibit a certain inhibitory effect on humanhepatocellular carcinoma cell line HepG2, human gastric carcinoma cell MGC-803, andhuman breast cancer cell line MCF-7, the highest inhibitory activity was showed on HepG2.The IC50values of two crude glycoproteins on HepG2were respectively36.794and15.164g/mL, the crude glycoprotein prepared by water extraction method exhibited the higheranticancer activity. Antioxidant activities of the two glycoproteins were evaluated by reducingpower, hydroxyl, superoxide anion and DPPH radical scavenging assay. The EC50values ofglycoprotein prepared by water extraction method on scavenging hydroxyl, superoxide anionand DPPH radical were2.771,2.051, and7.282mg/mL, respectively.Based on the anticancer and antioxidant activity test, a novel glycoprotein COGP2a withhighest anticancer and antioxidant activity was obtained from Camellia oleifera by waterextraction and purified by DEAE Sepharose F.F. ion exchage chromatography, SephadexG-100gel chromatography and AKTA protein purifier with SuperdexTMG-75. SDS-PAGE electrophoresis and HPGPC detection showed that COGP2a was a single band and a singlepeak, its weight-average molecular weight is25956Da. The thermal denaturation temperatureof COGP2a was64.7℃tested by differential scanning calorimetry (DSC), denaturationenthalpy value was120.6J/g. The specific rotation value of COGP2a was-32.5.The structure information of COGP2a was obtained from amino acid composition test,monosaccharide composition test, β-elimination reaction, Congo red test, infraredspectrometry, circular dichroism spectroscopy and LC-Q-TOF-MS-MS. The results showedthat the protein content of COGP2a was54.28%, the highest content of amino acid wasglutamic acid followed byarginine and aspartic acid. The sugar content of COGP2a was45.29%, the glycan of COGP2a mainly composed of rhamnose, arabinose, xylose, mannose,glucose, galactose with a molar ratio of1.29:1.65:5.81:1:16.15:14.47. The glycopeptidelinkage type of COGP2a was O-linked, the glycan of COGP2a was pyranose with-glycosidic bond structure. LC-Q-TOF-MS-MS analysis indicated that there were threematching peptide sequence, the m/z of which were1852.86,1304.63, and1574.67,respectively. Circular dichroismdata analysis showed that there was80.3%-helix,1.0%β-strand,6.4%β-turns and15.8%unordered in COGP2a.HepG2cells were selected as test subject to investigate the apoptosis effect of COGP2aon tumor cells. Results showed that the HepG2cells treated with COGP2a exhibited typicalapoptotic morphological character. Obvious nuclear chromosome condensation, nuclearfragmentation and strong blue fluorescence in HepG2cells stained by Hoechst33258wereobserved by fluorescence microscope. Scanning electron microscopy and transmissionelectron microscopy photos showed that the HepG2cells treated with COGP2a appeared toexhibit obvious nuclear condensation, nuclear fragmentation, abnormal organelles,disappearance of cell membrane surface microvilli and folds, large numbers of apoptoticbodies. Treated with COGP2a, The various stages of HepG2cells stained by PI and AnnexinV-FITC were analyzed by flow cytometry. COGP2a could open mitochondrial membranechannels and decrease mitochondrial membrane potential (ΔΨm) of HepG2cells stained byJC-1. Results also showed that the proportion of HepG2cells in G0/G1phase decreased, theratio of G2/M phase cells increased, HepG2cells were blocked into the next normal cellproliferation cycle. HepG2cells treated with COGP2a demonstrated an increasing expressionof Caspase-3protein observed by Western blotting, which suggested that COGP2a couldactivate the death receptor pathway.To evaluate the antitumor activity of COGP2a in vivo, liver cancer model wereestablished by transplanting H22hepatoma cells to the left oxter of mice. At the dosage of200mg/kg/d, COGP2a had strong anticancer activity with an inhibition rate of72.77%. Anincreased expression of the apoptotis-inducing protein Bax as compared to decreased expression of the apoptosis-inhibiting proteins Bcl-2in HepG2cells treated with COGP2awere observed by Western blotting. The histopathology of tumors tissue indicated that tumorcell treated with COGP2a dwindled in size and appeared nucleus pycnosis and necrosis. Inaddition, COGP2a were found to increase the mouse thymus and pancreas index, the numberof lymphocytes, white blood cell, the ratio of CD4/CD8and IFN-γ in different degree. Theseresults suggest that COGP2a can enhance the immune function of mice which contribute to itsantitumor effects.The D-galactose induced aging mice model was used to evaluate the antioxidant activityof COGP2a in vivo. Compared with the model group, COGP2a could significantly increasethe thymus and spleen indices of D-galactose induced aging mice (P<0.05). The result alsoshowed that compared with the model group, COGP2a could significantly enhanced theactivities of antioxidant enzymes (SOD and GSH-PX) and decreased the levels ofmalondialdehyde (MDA) and total carbonyl content in both serums and livers of aging mice(P<0.01). Statistical analysis showed that the correlation between the anti-tumor rate andantioxidant indices was statistically significant. These results suggest that COGP2a possesspotent antioxidant activities which contribute to its antitumor effects.