| Pine pollen contains a wide range of nutrition. There are multiple researches have been done on fatty acids and carbohydrates in Pine pollen, but the researches on proteins which has more than 10% content are very few.Proteins are the basic material of life, and peptides are a product of protein decomposition. Active peptides have many functions, such as, deduce blood pressure, anti-aging, antioxidant, anti-fatigue, enhance memory and promote the human body to absorb useful protein and many beneficial microelements. It has become a hot topic in the field of current bioengineering, so it is very worth to research. The purpose of this experiment is to extract and purify a kind of active peptide and research for its bioactivities, in order to make full use of Pine pollen.We use Piuns massoniana pollen as material, optimized every conditions through extraction time, the ratio of extraction material to liquid and PH. Peptide was extracted with salting out,dialysis, ion exchange chromatography, gel filtration, and high performance liquid chromatography, etc. Then determined the protein content and analyzed its effects of inhibiting HepG2 and stimulating the proliferation of spleen cells. The results are as follows:1,The protein from Piuns massoniana pollen mainly contains alkali-soluble protein to Glutenin, which accounted for 60% of total protein in pine pollen. This result is different from other pine pollen that protein is the main component in Piuns massoniana pollen. 2,Through the single-factor experimental analysis, we determined the best condition of extraction is : the ratio of extraction material to liquid was 1:20, the time of extraction was 4 hours, the frequency was 3, PH 8 and the temperature should be keeping at 4℃.3,In the process of ammonium sulfate fractional precipitation, after exploring various saturation, we determined three saturation, which are 0-60%,60-80% and 80-100%. The 0-60% component accounted for more than 50% of total precipitation.4,We obtained a pure peak that named for G2 after two Sephadex G25 chromatography and three peaks after semi-prepared HPLC. One of the peak G2H2 was more pure and it has two maximum absorption peak at 220nm and 280nm through spectrum scan. Besides, the G2H2 peak from G2H1, G2H2 and G2H3 also has a characteristic peptide absorption peak at 220nm and 280nm.5,After Sephadex G25 chromatography of the outside dialysis fluid, we obtained a peak of G10, which contained a large molecular weight. This protein showed two bands in SDS-PAGE, and its apparent molecular weight was 16KD.6,The purified component of G2H1, G10, G2H2, G2H3 could stimulate the proliferation of spleen lymphocytes at the concentration of 200ug/ml, the rate of promotion was respectively 29.81%, 46.10%, 39.73% and 14.84%. G2H1 had a terribly significant stimulation function (P<0.01) and G10 had a significant stimulation (P<0.05). These four components still had the function of promoting proliferation of spleen lymphocytes at the concentration of 100ug/ml. The rate was respectively 44.58%, 24.89%, 29.38% and 6.99%, of which G2H1 and G10 had a significant effect (P<0.05). The main peak G2 showed strong inhibition on HepG2 at both concentration of 100μg/ml and 200μg/ml, the rate was 36.34% and 47.69% (p<0.01).
|
PDF Full Text Request